Control of enzyme translocation in nanopore sequencing

ABSTRACT

The invention relates to devices and methods for nanopore sequencing. Methods for controlling translocation of through the nanopore are disclosed. The rate of transport of the template nucleic acids through the nanopore can be controlled using a translocating enzyme having two slow kinetic steps. The translocating enzyme and reaction conditions can be selected such that the translocating enzyme exhibits two kinetic steps wherein each of the kinetic steps has a rate constant, and the ratio of the rate constants of the kinetic steps is from 10:1 to 1:10. The invention also provides for using the signals from n-mers to provide sequence information, for example where the system has less than single base resolution. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/026,906, filed Sep. 13, 2013, which is acontinuation application of U.S. patent application Ser. No. 12/757,789filed Apr. 9, 2010, (now U.S. Pat. No. 8,986,928) which claims priorityto and benefit of U.S. Provisional Patent Application 61/168,431, filedApr. 10, 2009, the full disclosures of which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

The rapid determination of the nucleotide sequence of single- anddouble-stranded DNA and RNA is a major goal of researchers seeking toobtain the sequence for the entire genome of an organism. The ability todetermine the sequence of nucleic acids in DNA or RNA has additionalimportance in identifying genetic mutations and polymorphisms. Theconcept of using nanometer-sized holes, or “nanopores,” to characterizebiological macromolecules and polymer molecules has recently beendeveloped.

Nanopore-based analysis methods often involve passing a polymericmolecule, for example single-stranded DNA (“ssDNA”), through ananoscopic opening while monitoring a signal such as an electricalsignal. Typically, the nanopore is designed to have a size that allowsthe polymer to pass only in a sequential, single file order. As thepolymer molecule passes through the nanopore, differences in thechemical and physical properties of the monomeric units that make up thepolymer, for example, the nucleotides that compose the ssDNA, aretranslated into characteristic electrical signals.

The signal can, for example, be detected as a modulation of the ioniccurrent by the passage of a DNA molecule through the nanopore, whichcurrent is created by an applied voltage across the nanopore-bearingmembrane or film. Because of structural differences between differentnucleotides, different types of nucleotides interrupt the current indifferent ways, with each different type of nucleotide within the ssDNAproducing a type-specific modulation in the current as it passes througha nanopore, and thus allowing the sequence of the DNA to be determined.

Nanopores that have been used for sequencing DNA include proteinnanopores held within lipid bilayer membranes, such as α-hemolysinnanopores, and solid state nanopores formed, for example, by ion beamsculpting of a solid state thin film. Devices using nanopores tosequence DNA and RNA molecules have generally not been capable ofreading sequence at a single-nucleotide resolution.

While this prior work has shown the promise of nanopores for detectingsome sequence information, there is a need for accurate, reliabledevices and methods for measuring sequences such as those of RNA andDNA. Accordingly, there is a need for a method of fabricating arrays ofnanopores in a form that is amenable to manufacturing. Similarly, thereis also a related need for devices capable of sequencing moleculeshaving nanoscale dimensions at a high speed and at a high level ofresolution.

SUMMARY OF THE INVENTION

In some aspects, the invention provides a device for determining polymersequence information comprising: a substrate comprising an array ofnanopores; each nanopore fluidically connected to an upper fluidicregion and a lower fluidic region; wherein each upper fluidic region isfluidically connected through an upper resistive opening to an upperliquid volume. In some embodiments the upper liquid volume isfluidically connected to two or more upper fluidic regions. In someembodiments each lower fluidic region is fluidically connected through alower resistive opening to a lower liquid volume, and wherein the lowerliquid volume is fluidically connected to two or more lower fluidicregions.

In some embodiments the substrate is a semiconductor comprising circuitelements. In some embodiments either the upper fluidic region or thelower fluidic region for each nanopore or both the lower fluidic regionand the upper fluidic region for each nanopore is electrically connectedto a circuit element. In some embodiments the circuit element comprisesan amplifier, an analog-to-digital converter, or a clock circuit.

In some embodiments the resistive opening comprises one or morechannels. In some embodiments the length and width of the one or morechannels are selected to provide a suitable resistance drop across theresistive opening. In some embodiments the conduit is a channel througha polymeric layer. In some embodiments the polymeric layer ispolydimethylsiloxane (PDMS).

In some embodiments the device further comprises an upper driveelectrode in the upper liquid volume, a lower drive electrode in thelower liquid volume, and a measurement electrode in either the upperliquid volume or the lower liquid volume.

In some embodiments the device further comprises an upper driveelectrode in the upper liquid volume, a lower drive electrode in thelower liquid volume, and an upper measurement electrode in the upperliquid volume and a lower measurement electrode in the lower liquidvolume.

In some embodiments the nanopore, upper fluidic reservoir and lowerfluidic reservoir are disposed within a channel that extends through thesubstrate. In some embodiments the upper fluidic reservoir and lowerfluidic reservoir each open to the same side of the substrate.

In some aspects, the invention provides a polymer sequencing devicecomprising: a) a nanopore layer comprising an array of nanopores, eachnanopore having a cross sectional dimension of 1 to 10 nanometers, andhaving a top and a bottom opening, wherein the bottom opening of eachnanopore opens into a discrete reservoir, resulting in an array ofreservoirs, wherein each reservoir comprises one or more electrodes, thenanopore layer physically and electrically connected to a semiconductorchip, and b) the semiconductor chip, comprising an array of circuitelements, wherein each of the electrodes in the array of reservoirs isconnected to at least one circuit element on the semiconductor chip.

In some embodiments the array of nanopores comprises an array of holesin a solid substrate, each hole comprising a protein nanopore. In someembodiments each protein nanopore is held in place in its hole with alipid bilayer. In some embodiments the top opening of the nanopores openinto an upper reservoir. In some embodiments the circuit elementscomprise amplifiers, analog to digital converters, or clock circuits.

In some aspects, the invention provides a method of fabricating apolymer sequencing device comprising: a) obtaining a semiconductorsubstrate; b) processing the semiconductor substrate to create an arrayof microfluidic features, wherein the microfluidic features are capableof supporting an array of nanopores; c) subsequently producing circuitelements on the substrate that are electronically coupled to themicrofluidic features; and d) introducing nanopores into themicrofluidic features.

In some embodiments the circuit elements are CMOS circuit elements. Insome embodiments the CMOS circuit elements comprise amplifiers, analogto digital converters.

In some aspects, the invention provides a method of fabricating apolymer sequencing device comprising the following steps in the orderpresented: a) obtaining a semiconductor substrate; b) processing thesemiconductor substrate to create an array of CMOS circuits, withoutcarrying out an aluminum deposition step; c) processing thesemiconductor substrate having the CMOS circuits to produce microfluidicfeatures, wherein the microfluidic features are capable of supportingnanopores; d) subsequently performing an aluminum deposition step tocreate conductive features; and e) introducing nanopores into themicrofluidic features.

In some embodiments the processing of step (c) to create themicrofluidic features subjects the semiconductor substrate totemperatures greater than about 250° C.

In some aspects, the invention provides a method for fabricating apolymer sequencing device comprising: a) producing an insulator layerhaving microfluidic elements comprising an array of pores extendingthrough the insulator; b) bonding the insulator layer with asemiconductor layer; c) exposing the semiconducting layer to etchantthrough the pores in the insulator layer to produce discrete reservoirsin the semiconductor layer; d) removing portions of the semiconductorlayer to isolate the discrete reservoirs from one another, e)incorporating electrical contacts into the semiconductor layer thatallow current to be directed to each of the discrete reservoirs; and f)bonding an electric circuit layer to the semiconducting layer such thatthe electric circuits on the electric circuit layer are electricallyconnected to the electrical contacts on the semiconductor layer.

In some embodiments the method further comprises the step of addingnanopores into each of the pores.

In some embodiments the method further comprises two or more electrodeswithin each of the discrete reservoirs.

In some aspects, the invention provides a method for fabricating apolymer sequencing device comprising: a) producing an insulator layerhaving microfluidic elements comprising an array of pores extendingthrough the insulator; b) bonding the insulator layer with asemiconductor layer wherein the semiconducting layer comprises an arrayof wells corresponding to the pores on the insulator layer, whereby thebonding produces an array of discrete reservoirs, each discretereservoir connected to a pore; c) removing portions of the semiconductorlayer to isolate the discrete reservoirs from one another d) addingelectrical contacts to the semiconductor layer that allow current to bedirected to each of the discrete reservoirs; and e) bonding an electriccircuit layer to the semiconducting layer such that the electriccircuits on the electric circuit layer are electrically connected to theelectrical contacts on the semiconductor layer.

In some aspects, the invention provides a method for fabricating apolymer sequencing device comprising: a) obtaining an SOI substratecomprising a top silicon layer, an insulator layer, and a bottom siliconlayer; b) processing the top silicon layer and bottom silicon layer toremove portions of each layer to produce an array of exposed regions ofthe insulator layer in which both the top and bottom surfaces of theinsulator layer are exposed; c) processing the top silicon layer or thebottom silicon layer or both the top silicon layer and bottom siliconlayer to add electrodes and electrical circuits; and d) processing theinsulator layer to produce an array of pores through the exposed regionsof the insulator layer.

In some embodiments the method further comprises adding polymer layersto the top of the device, the bottom of the device, or to the top and tothe bottom of the device to produce microfluidic features.

In some embodiments the method further comprises inserting a nanoporeinto the pores in the insulator layer.

In some aspects, the invention provides a method for determiningsequence information about a polymer molecule comprising: a) providing adevice comprising a substrate having an array of nanopores; eachnanopore fluidically connected to an upper fluidic region and a lowerfluidic region; wherein each upper fluidic region is fluidicallyconnected through a an upper resistive opening to an upper liquidvolume; and each lower fluidic region is connected to a lower liquidvolume, and wherein the upper liquid volume and the lower liquid volumeare each fluidically connected to two or more fluidic regions, whereinthe device comprises an upper drive electrode in the upper liquidvolume, a lower drive electrode in the lower liquid volume, and ameasurement electrode in either the upper liquid volume or the lowerliquid volume; b) placing a polymer molecule to be sequenced into one ormore upper fluidic regions; c) applying a voltage across the upper andlower drive electrodes so as to pass a current through the nanopore suchthat the polymer molecule is translated through the nanopore; d)measuring the current through the nanopore over time; and e) using themeasured current over time in step (d) to determine sequence informationabout the polymer molecule.

In some embodiments the substrate comprises electronic circuitselectrically coupled to the measurement electrodes which at leastpartially process signals from the measurement electrodes.

In some embodiments the upper drive electrode and lower drive electrodeare each biased to a voltage above or below ground, and at least aportion of the substrate electrically connected to the electroniccircuits is held at ground potential.

In some aspects, the invention provides a method for determiningsequence information about a polymer molecule comprising: a) providing adevice having an array of nanopores, each connected to upper and lowerfluid regions; wherein the device comprises electronic circuitselectrically connected to electrodes in either the upper fluid regionsor lower fluid regions or both the upper and lower fluid regions; b)placing a polymer molecule in an upper fluid region; c) applying avoltage across the nanopore whereby the polymer molecule is translocatedthrough the nanopore; d) using the electronic circuits to monitor thecurrent through the nanopore over time, wherein the electronic circuitsprocess the incoming current over time to record events, therebygenerating event data; and e) using the event data from step (d) toobtain sequence information about the polymer molecule.

In some embodiments the events comprise a change in current level aboveor below a specified threshold. In some embodiments the electroniccircuit records the events, the average current before the events andthe average current after the events. In some embodiments the event datais generated without reference to time.

In some embodiments a clock circuit is used such that the relative timethat the events occurred is also determined. In some embodiments theevent data generated by the electronic circuits on the device istransmitted from the device for further processing. In some embodimentsthe information is transmitted optically.

In some aspects, the invention provides a method for determining thesequence of a polymer having two or more types of monomeric units in asolution comprising: a) actively translocating the polymer through apore; b) measuring a property which has a value that varies depending onwhether and which of the two or more a types of monomeric unit is in thepore, wherein the measuring is performed as a function of time while thepolymer is actively translocating; and c) determining the sequence ofthe two or more types of monomeric units in the polymer using themeasured property from step (b) by performing a process including thesteps of: (i) deconvolution, (ii) peak finding, and (iii) peakclassification.

In some embodiments the polymer is a nucleic acid, the monomeric unitsare nucleotide bases or nucleotide analogs, and the measured property iscurrent. In some embodiments the deconvolution comprises (a) carryingout measurements of current as a function of time on nucleic acidshaving known sequences to produce calibration information, and (b) usingthe calibration information perform the deconvolution. In someembodiments the deconvolution uses a Weiner, Jansson, or Richardson-Levydeconvolution. In some embodiments the peak classification is performedby a heuristic tree algorithm, Bayesian network, hidden Markov model, orconditional random field. In some embodiments the method furthercomprises step (iv) of quality estimation.

In some embodiments the measurements on nucleic acids having knownsequences comprising known n-mers. In some embodiments the known n-mersare 3-mers, 4-mers, 5-mers or 6-mers.

DESCRIPTION OF THE FIGURES

FIG. 1A shows an embodiment of an array or nanopores of the inventionhaving resistive openings and incorporated electronics associated withthe nanopores.

FIG. 1B shows an alternative embodiment wherein the input and outputpores from the nanopore extend to the same surface.

FIG. 2 shows a structure of the invention comprising resistive openings.

FIG. 3 shows a cross sectional view of an embodiment of a multiplexnanopore sequencing device of the invention having discrete reservoirs.

FIG. 4 shows an embodiment of the invention comprising a salt bridge.

FIG. 5 shows an embodiment of the invention illustrating the chemistryused to produce an array of hybrid nanopores of the invention.

FIG. 6 shows a process of the invention wherein a nanopore/electrode isproduced with a self-aligned etching process.

FIG. 7 shows the production of microfluidic features in a semiconductorsubstrate prior to wafer bonding.

FIG. 8 shows a schematic for a process for producing nanopore arraysusing an SOI wafer.

FIG. 9 illustrates how polymers such as PDMS can be used to fluidicallyseal portions of the device.

FIG. 10 shows the passage of DNA or RNA translocating under an appliedvoltage though a nanopore structure within a physical barrier.

FIG. 11 shows the passage of DNA or RNA translocating under an appliedvoltage though a nanopore structure within a physical barrier where thebarrier comprise DNA binding proteins.

FIG. 12 shows an embodiment for controlling translocation duringsequencing in which a DNA polymerase enzyme with strand displacement isused to create a single strand of DNA which is then translocated throughthe nanopore.

FIG. 13 shows an embodiment for determining sequence information about atemplate polymer by controlling translocation.

FIG. 14 illustrates electrical control of translocation of a moleculethrough a nanopore.

FIGS. 15A and 15B illustrate the use of a molecular brake to controltranslocation through the membrane.

FIG. 16 shows a process for producing a molecular brake.

FIG. 17 illustrates nanopores having different profiles.

FIG. 18 illustrates transporting a polymer through a nanopore usingalternating fields.

FIG. 19 shows a structure with multiple layers of conducting pads thatare electrically isolated and individually addressable.

FIG. 20 illustrates a molecular pawl.

FIG. 21 shows a multi-pawl aperture.

FIG. 22 shows a structure for multiple stage nanopore sequencing.

FIG. 23A shows a schematic drawing of a multi-staged tunneling currentmeasurement system. FIG. 23B shows an alternative multi-stage tunnelingembodiment having one channel with several transverse tunnelingmeasurement stages.

FIGS. 24A and 24B illustrate a nanopore is depressed within a well.

FIGS. 25A-25D each show a protein nanopore that has a linker molecule toattach DNA.

FIG. 26 shows a method for multi-pass sequencing.

FIGS. 27A and 27B show drawing the DNA back and forth, while it isretained by the pore.

FIG. 28 shows current levels corresponding to different portions of aDNA strand passing through a nanopore.

FIG. 29 shows an algorithm for using a lookup table for base calling.

FIG. 30 provides a flow chart illustrating dynamic interventionalnanopore sequencing.

FIGS. 31A-31D show the use of tethered magnetic particles to control DNAtranslocation through the pore.

FIG. 32 is a schematic illustration of the reaction cycle forpolymerase-mediated nucleic acid primer extension.

FIG. 33 shows a theoretical representation of the probability densityfor residence time for a polymerase reaction having 1 rate-limiting stepor two rate-limiting steps within an observable phase.

DETAILED DESCRIPTION OF THE INVENTION

I. General

The invention relates to devices, systems, and methods for sequencingpolymers using nanopores. In particular, the invention relates tomultiplex sequencing in which sequencing data is simultaneously obtainedfrom multiple nanopores. In some aspects, the invention relates tomultiplex nanopore sequencing devices that directly incorporatesemiconductor devices, such as CMOS devices. The devices of theinvention can be made wherein the nanopores are formed in asemiconductor substrate, such as silicon. Alternatively, the divides canbe made in a composite semiconductor substrate such assilicon-insulator-silicon (SOI), or can be made by bonding togethersemiconductor and insulator components.

The incorporation of semiconductors such as silicon into the devicesprovides for the inclusion of electronic circuitry in close associationwith the nanopores. For example, the use of silicon allows for amultiplex device having an array of electronic circuits wherein eachnanopore in the array is directly associated with a set of electroniccircuits. These circuits can provide the functions of measurement, datamanipulation, data storage, and data transfer. The circuits can provideamplification, analog to digital conversion, signal processing, memory,and data output.

In some aspects, the invention relates to devices and methods whichallow for multiplex electronic sequencing measurements in a manner thatreduces or eliminates cross-talk between the nanopores in the nanoporearray. In some cases it is desirable for a nanopore sequencingmeasurement system to have a pair of drive electrodes that drive currentthrough the nanopores, and one or more measurement electrodes thatmeasure the current through the nanopore. It can be desirable to havethe drive electrodes drive current through multiple nanopores in thenanopore array, and have measurement electrodes that are directlyassociated with each nanopore. We have found that this type of systemcan be obtained by the incorporation of resistive openings, whichconnect a reservoir of fluid in contact with the nanopore to a volume offluid in contact with a drive electrode in a manner that creates aresistive drop across the resistive opening, but allows for fluidicconnection and for ion transport between the reservoir of fluid incontact with the nanopore and the volume of fluid in contact with thedrive electrode.

The resistive opening can be made from any suitable structure thatprovides for a resistive drop across two fluid regions while allowingfor the passage of fluid including ions between the fluid regions. Ingeneral, the resistive opening will impede, but not prevent the flow ofions. The resistive opening can comprise, for example, one or morenarrow holes, apertures, or conduits. The resistive opening can comprisea porous or fibrous structure such as a nanoporous or nanofibermaterial. The resistive opening can comprise a single, or multiple,long, narrow channels. Such channels can be formed, for example, in apolymeric material such as polydimethylsiloxane (PDMS).

The nanopore sequencing of the invention relates to the sequencing ofpolymers. The polymers to be sequenced can be, for example, nucleicacids such as RNA or DNA, proteins, polypeptides, polysaccharides, orother polymers for which information about the sequence is of value. Insome embodiments, the sequencing is performed by measuring themodulation of current as the polymer molecule, e.g. a single-strandedDNA molecule passes through the nanopore. In some cases, the polymer asa whole does not pass through the pore, but portions of the polymer, ormolecules associated with portions of the polymer pass through thenanopore, and are detected. For example, in some cases, a nucleic acidis sequentially degraded, sequentially releasing monomeric units, e.g.by an exonuclease, and the monomeric units are detected as they passthrough the nanopore. Certain aspects and embodiments are described asbeing implemented with specific materials, e.g. a specific polymer. Itunderstood that the embodiments described can be implemented using anysuitable material such as those described elsewhere herein or as knownin the art.

II. Nanopore Sequencing Devices

The invention relates in some aspects to devices for multiplex nanoporesequencing. In some cases, the devices of the invention compriseresistive openings between fluid regions in contact with the nanoporeand fluid regions which house a drive electrode. The devices of theinvention can be made using a semiconductor substrate such as silicon toallow for incorporated electronic circuitry to be located near each ofthe nanopores or nanometer scale apertures in the array of nanoporeswhich comprise the multiplex sequencing device. The devices of theinvention will therefore comprise arrays of both microfluidic andelectronic elements. In some cases, the semiconductor which has theelectronic elements also includes microfluidic elements that contain thenanopores. In some cases, the semiconductor having the electronicelements is bonded to another layer which has incorporated microfluidicelements that contain the nanopores.

The devices of the invention generally comprise a microfluidic elementinto which a nanopore is disposed. This microfluidic element willgenerally provide for fluid regions on either side of the nanoporethrough which the molecules to be detected for sequence determinationwill pass. In some cases, the fluid regions on either side of thenanopore are referred to as the cis and trans regions, where themolecule to be measured generally travels from the cis region to thetrans region through the nanopore. For the purposes of description, wesometimes use the terms upper and lower to describe such reservoirs andother fluid regions. It is to be understood that the terms upper andlower are used as relative rather than absolute terms, and in somecases, the upper and lower regions may be in the same plane of thedevice. The upper and lower fluidic regions are electrically connectedeither by direct contact, or by fluidic (ionic) contact with drive andmeasurement electrodes. In some cases, the upper and lower fluid regionsextend through a substrate, in other cases, the upper and lower fluidregions are disposed within a layer, for example, where both the upperand lower fluidic regions open to the same surface of a substrate.Methods for semiconductor and microfluidic fabrication described hereinand as known in the art can be employed to fabricate the devices of theinvention.

FIG. 1A shows a cross section of an exemplary multiplex nanoporesequencing device of the invention comprising resistive openings.Substrate layer 100 comprises a semiconductor material such as silicon.The semiconductor substrate comprises an array of holes or porescomprising nanopores. FIG. 1A shows two pores. Devices of the inventioncan have any suitable number of pores to facilitate multiplexsequencing, for example 2 to 10 pores, 10 to 100 pores, 100 to 1000pores, 1000 to 10,000 pores or more than 10,000 pores. Each of the poreshas a nanopore or nanometer scale aperture 150. As used herein the termnanopore, nanometer scale aperture, and nanoscale aperture are usedinterchangeably. In each case, the term refers to an opening which is ofa size such that when molecules of interest pass through the opening,the passage of the molecules can be detected by a change in signal, forexample, electrical signal, e.g. current. In some cases the nanoporecomprises a protein, such as alpha-hemolysin or MspA, which can bemodified or unmodified. In some cases, the nanopore is disposed within amembrane, or lipid bilayer, which can be attached to the surface of themicrofluidic region of the device of the invention by using surfacetreatments as described herein and as known in the art. In some cases,the nanopore can be a solid state nanopore. Solid state nanopores can beproduced as described in U.S. Pat. No. 7,258,838, U.S. Pat. No.7,504,058 In some cases, the nanopore comprises a hybrid protein/solidstate nanopore in which a nanopore protein is incorporated into a solidstate nanopore.

The device of FIG. 1A has upper fluidic region 130 and lower fluidicregion 140, which are in contact with the nanopore 150. Upper fluidicregion 130 is fluidically connected to upper fluid volume 160 throughthe upper resistive opening 120. In addition, in this device, lowerfluidic region 130 is fluidically connected to lower fluid volume 170through the lower resistive opening 110. Generally, the drive electrodeswill be disposed in fluid volumes 160 and 170. The fluid volumes 160 and170 can be in fluidic contact with multiple pores in the substrate 100containing nanopores. The resistive opening minimizes the electricalcrosstalk between the multiplex pores in the device. The semiconductorsubstrate 100 also comprises electrical circuits 180 and 185. Suchcircuits can be used to measure, process, and store electronic data andsignals related to the sequencing measurements. For example, thecircuits can be connected to measurement electrodes extending into theupper fluid region 130 and/or lower fluid region 140 to measure signalsassociated with nanopore 150. In some cases, each nanopore will have aset of embedded circuitry associated with it, for example as shown wherecircuitry 185 is used to measure and process electrical characteristicsrelated to nanopore 155. The electronic circuits can be made by anysuitable semiconductor processing technique described herein or known inthe art. In some cases the circuits comprise CMOS circuits. Thenanopores can be any suitable nanopore including a solid state nanopore,a protein nanopore, or a hybrid protein/solid state nanopore. Thenanopores illustrated in FIG. 1A comprise hybrid nanopores, described inmore detail below, in which a solid state nanopore is sized toaccommodate a single nanopore protein, and the surface of the apertureis modified in order to hold the nanopore protein in place.

FIG. 1B shows a cross sectional view of an alternative embodiment of ananopore in an array of nanopores in which the upper fluidic region 230and the lower fluidic region 240 each open to the top surface of siliconsubstrate 200 through resistive openings 220 and 210 to contact upperfluid volume 270 and lower fluid volume 260. As described above, thefluid volumes 260 and 270 can house the drive electrodes. The fluidvolumes 260 and 270 can extend across multiple nanopores in thesubstrate. The semiconductor substrate 200 comprises electronic circuits280 which can be electronically connected to measurement electrodes asdescribed above. FIG. 1B shows one nanopore and surrounding microfluidicand electronic structures. The device of the invention will generallycomprise an array of hundreds to thousands or more of such structures.

In some cases herein the term “each” is used when referring to themicrofluidic or electronic elements in an array on the device. Ingeneral, the term each, does not mean all. For example, an array inwhich each microfluidic element comprises a nanopore may include anarray in which a subset of all of the microfluidic elements comprise ananopore. The meaning of the term “each” as used herein should beunderstood in light of the context in which the term is used.

In some embodiments the devices comprise an nanopore layer is separatefrom the semiconductor layer comprising the circuitry. In such cases,the substrate comprising the nanopore layer is typically electricallyinsulating. The substrate can be made from any suitable materialincluding, for example, polymers, oxides, such as silicon oxide, anitride, or can be made from a semiconductor material such as silicon.

One aspect of the invention is the incorporation of resistive openingsinto these structures for facilitating the use of a single driveelectrode for multiple nanopores (a constriction architecture).

The incorporation of resistive openings associated with each nanoporecan be useful for multiplexing and miniaturizing a system for nanoporeDNA sequencing, providing for the use of a single drive electrode toprovide the applied potential for each of the in-parallel nanopores. Theuse of a single set of drive electrodes can be advantageous because itsimplifies the electronics and enables one to place the drive electrodeaway from the individual pores so that bubble-formation due toelectrolysis at the electrode will not disrupt the nanopore orsupporting lipid bilayer, and such that chemical species generated atthe drive electrodes, for example acids, bases, oxidizing, and reducingspecies do not interfere with the sequencing measurements. With one setof drive electrodes, each nanopore generally requires one or moremeasurement electrodes. However, with one set of drive electrodes, therecan be cross-talk between adjacent nanopores. For example, at any givenmoment, some pores will be open and others will be closed. This canresult in statistical fluctuation of the resistance across the totalcircuit over time, which can lead to errors in determining polymersequence.

In some aspects of this invention, a single drive voltage source canused for all the nanopores, and each nanopore is protected by aconstriction (resistive opening). FIG. 2 shows an arrangement in whichconstrictions in the substrate act to electrically isolate it from thefluctuations described above. In some cases, there is a constriction, orresistive opening only above or only below the nanopore. In some casesthere is a constriction, or resistive opening both above and below thenanopore. The resistive openings create a resistance drop between thefluid regions that they span. The resistance drop across a resistiveopening is generally on the same order as the resistance drop across thenanopore and is generally equal to or lower than the resistive dropacross the nanopore. In some cases the resistance drop across theresistive opening is about 1 K-ohm to about 100 G-ohm, from about 1M-ohm to about 10 G-ohm. In some cases, the resistance drop is about thesame as the resistance drop across an unblocked pore. In some cases, theresistance drop across the resistive opening is lower by a factor ofgreater than about 5, 10, 20, 50 or 100 relative to the resistanceacross an unblocked pore. In other cases, the resistance drop across theresistive opening is higher by a factor greater than about 5, 10, 20, 50or 100 relative to the resistance across an unblocked pore.

In some aspects, the invention relates to devices and methods whichallow for multiplex electronic sequencing measurements in a manner thatreduces or eliminates cross-talk between the nanopores in the nanoporearray. In some cases it is desirable for a nanopore sequencingmeasurement system to have a pair of drive electrodes that drive currentthrough the nanopores, and one or more measurement electrodes thatmeasure the current through the nanopore. It can be desirable to havethe drive electrodes drive current through multiple nanopores in thenanopore array, and have measurement electrodes that are directlyassociated with each nanopore. We have found that this type of systemcan be obtained by the incorporation of resistive openings, whichconnect a reservoir of fluid in contact with the nanopore to a volume offluid in contact with a drive electrode in a manner that creates aresistive drop across the resistive opening, but allows for fluidicconnection and for ion transport between the reservoir of fluid incontact with the nanopore and the volume of fluid in contact with thedrive electrode.

These resistive openings can be optimized for several type of operatingconditions. For example, in some embodiments it is convenient for theresistive opening to act as a reference resistor, and in some cases itis desirable to have this resistance be well balanced with thesequencing nanopore resistance. One means of attaining this is for theresistive opening to comprise an additional nanopore identical to thesequencing nanopore. In this way the balance between the referenceresistive opening and the sequencing nanopore is automatically optimal.In other embodiments it is desirable to minimize the stray seriescapacitance of the system, and in these cases a low capacitance can beachieved by increasing the thickness of the membrane while at the sametime increasing the cross-sectional area of the aperture of theresistive opening. In some embodiments this membrane could be 2 timesthe thickness of the sequencing nanopore membrane, in still others, itcould be 10, 30, 100, 300, 1000, 3000 or 10000 times thicker than thesequencing membrane. It is also of interest that the reference resistiveopening be fabricated in a membrane that has a small surface area, ascapacitance is typically proportional to surface area. In someembodiments, the reference resistive opening is 10 microns in diameter,in others it is 3 microns in diameter, in others it is 1 micron indiameter. In others there is no membrane and only a resistive opening inan otherwise solid structure.

The effect of a series of resistive openings can be simulated, forexample, using a program such as Matlab. Such simulations have been usedto demonstrates the ratio of the mean resistance in such a circuit tothe standard deviation of the resistance, given N nanopores in parallel,a probability P of each nanopore being open (derived from the duty cycleof current blockage due to passing nucleotides to be ˜ 1/30), andassuming typical resistance values for open and closed nanopores, JACS,128:1705-1710 (2006). For example, a simulation showed that for N=10nanopores, one could incorporate a constriction resistance R1 of ≧5e9ohms for the standard deviation of the resistance to be < 1/100 of themean resistance. Such a resistance could be accomplished, for example,by placing another protein nanopore within a lipid bilayer in theconstriction, by having the constriction comprise an opening of ˜2-3 nmdiameter and 1 nm deep opening, or by using a larger diameterconstriction that is deeper than 1 nm. This level of resistance couldalso be accomplished using nanoporous or fibrous materials.Alternatively, a long narrow channel, e.g. a channel through a polymersuch as PDMS can provide a resistive opening. The long narrow channelcan have a cross-sectional dimension of about 3 nm to about a micrometerand have an aspect ratio of 1:5, 1:10, 1:100, 1:1000, 1:10,000 or more.Another advantage of the use of a resistive opening is that it can helpprevent crosstalk of chemical species between nanopores. For example,resistive openings can prevent exonuclease-excised nucleotides fromdiffusing into an unwanted nanopore.

In one aspect, the invention comprises a device for determining polymersequence information comprising: a substrate comprising an array ofnanopores; each nanopore fluidically connected to an upper fluidicregion and a lower fluidic region; wherein each upper fluidic region isfluidically connected through a resistive opening to an upper liquidvolume, wherein the upper liquid volume is fluidically connected to twoor more upper fluidic regions.

In some case each lower fluidic region is fluidically connected througha resistive opening to a lower liquid volume, and wherein the lowerliquid volume is fluidically connected to two or more lower fluidicregions. In some embodiments the substrate is a semiconductor comprisingcircuit elements. In some embodiments, either the upper fluidic regionor the lower fluidic region for each nanopore or both the lower fluidicregion and the upper fluidic region for each nanopore is electricallyconnected to a circuit element. In some embodiments the circuit elementcomprises an amplifier, an analog-to-digital converter, or a clockcircuit. In some embodiments the resistive opening comprises one or morechannels. In some embodiments the length and width of the one or morechannels are selected to provide a suitable resistance drop across theresistive opening. In some embodiments the conduit is a channel througha polymeric layer. In some embodiments the polymeric layer ispolydimethylsiloxane (PDMS).

The devices of the invention can also include an upper drive electrodein the upper liquid volume, a lower drive electrode in the lower liquidvolume, and a measurement electrode in either the upper liquid volume orthe lower liquid volume. Alternatively, the devices can include an upperdrive electrode in the upper liquid volume, a lower drive electrode inthe lower liquid volume, and an upper measurement electrode in the upperliquid volume and a lower measurement electrode in the lower liquidvolume.

In some cases, the nanopore, upper fluidic reservoir and lower fluidicreservoir are disposed within a channel that extends through thesubstrate. In some cases the upper fluidic reservoir and lower fluidicreservoir each open to the same side of the substrate.

In some embodiments, the devices of the invention do not compriseresistive openings.

In some embodiments, the devices comprise discrete reservoirs, whereineach discrete reservoir is associated with one nanopore. In some casesthe discrete reservoir can be connected to an upper fluidic region, alower fluidic region, or both an upper and lower fluidic region of thenanopore. In other cases, the discrete fluidic regions for each nanoporeare separated, such that there is no fluidic contact between theregions. FIG. 3 shows a cross sectional view of an embodiment of amultiplex nanopore sequencing device of the invention having discretereservoirs. The device has an array of pores 320 which hold nanopores350. As shown, nanopore 350 is disposed at the base of the pore 320. Inother embodiments, it could be placed in any other suitable portion ofthe pore 320 including at or near the top or in the middle region. Thenanopores 350 can comprise either solid state nanopores, proteinnanopores, or hybrid nanopores such as those described herein. Thepores, 320 are in fluidic contact with discrete reservoirs 310 below,and in this embodiment with upper fluid volume 360. In otherembodiments, the upper fluidic region can also be a discrete region,associated only with that nanopore. For example, the top surface of thedevice can have separate wells isolating the pores, or can havehydrophobic barriers between the pores allowing for separate fluidicregions, each associated with one pore. Where each pore has a distinctfluidic region, the drive voltage for transporting the molecules throughthe pores is supplied to each separate nanopore. The discrete fluidicreservoirs are each connected to electrodes 340 for providing drivecurrent and for measuring electrical properties for sequencedetermination. In some cases, the electrodes 340 will comprise twoelectrodes to each discrete reservoir, one to act as a drive electrode,and the other to act as a measurement electrode. In some cases, theinner surface of the discrete reservoir 310 can have a high conductivityelectrode such as gold, platinum, or aluminum. In some cases, theelectrode can be coated with a dielectric material such as a low Kdielectric. The electrodes 340 can be connected to electronic circuitry380, which can include, for example, amplifiers for amplifying themeasured electrical signal. The electronic circuitry can be produced,for example in a semiconductor substrate 390. A device such as thatshown in FIG. 3 can be produced using flip chip methods. FIG. 3 shows 5pores 320 having nanopores 350, but such a device of the invention mayhave more or fewer nanopores as described herein. The devices may have10s to 100s to 1000s of pores. The pores can be arranged linearly, or ina two dimensional array structure.

The discrete fluid reservoirs can be of any suitable shape and suitablevolume. The dimensions of the discrete reservoirs will generally be onthe order of a micron, 10 microns, or 100s of microns.

One aspect of the invention is a polymer sequencing device comprising: ananopore layer comprising an array of nanopores, each nanopore having across sectional dimension of about 1 to 10 nanometers, and having a topand a bottom opening, wherein the bottom opening of each nanopore opensinto a discrete reservoir, resulting in an array of reservoirs, whereineach reservoir comprises one or more electrodes; and a semiconductorchip, comprising an array of circuit elements, wherein each of theelectrodes in the array of reservoirs is connected to at least onecircuit element on the semiconductor chip.

In some embodiments the array of nanopores comprises an array of holesin a solid substrate, each hole comprising a protein nanopore. In someembodiments each protein nanopore is held in place in its hole with alipid bilayer.

In some cases the top opening of the nanopores open into an upperreservoir. In some cases the circuit elements comprise amplifiers,analog to digital converters, or clock circuits.

In some embodiments the devices of the invention comprise a salt bridgewhich can be use to isolate liquid regions in the device. For example, asalt bridge can be used in order to provide for one buffer suited forbiochemistry, and another suited for electrical measurement. The saltbridge isolation can also prevent sensitive reagents from undergoingelectrochemical reactions at the electrodes, which can occur for somecompounds at even low voltages. In some cases porous materials, likelow-k dielectrics can be used. For example, a salt bridge can beincorporated between a chamber where the nanopore is held, and a chamberwhere the drive voltage and the resulting currents are measure. The saltbridge allows for the composition of each solution to be optimized toprovide ideal biochemical behavior and ideal electrical measurementsomewhat separately. FIG. 4 shows an embodiment comprising a saltbridge. In this embodiment, a biological buffer is in the fluid regionsthat are in direct contact with the protein nanopore. A salt bridgeprovides an ionic connection between the biological buffer and a fluidregion having a measurement buffer. The fluid region comprises anelectrode which acts as a drive electrode, and in some cases also actsas a measurement electrode.

In some embodiments, the devices utilize MESA structures. Thesestructures can be used, for example, when building electrical cellsstraight onto either a silicon or an SOI wafer. The MESA designs asknown in the CMOS industry can be used to guarantee insulation of thedifferent cells in the device. See, e.g. U.S. Pat. No. 5,049,513.

Hybrid Nanopores—Surface Functionalization

One aspect of the invention is the use of a hybrid solid state-proteinnanopore in the multiplexed nanopore sequencing device. We describeherein methods for functionalizing a solid-state pore either to enhanceits ability to detect or sequence a polymer such as DNA, or to enablehybrid protein/solid state nanopore.

Two approaches are typically used for nanopore polymer (DNA) sequencing:the first uses a protein nanopore (e.g. alpha-hemolysin, or MspA)embedded in a lipid membrane, and the second uses a solid-statenanopore. Protein nanopores have the advantage that as biomolecule, theyself-assemble and are all identical to one another. In addition, it ispossible to genetically engineer them to confer desired attributes or tocreate a fusion protein (e.g. an exonuclease+alpha-hemolysin). On theother hand, solid state nanopores have the advantage that they are morerobust and stable compared to a protein embedded in a lipid membrane.Furthermore, solid state nanopores can in some cases be multiplexed andbatch fabricated in an efficient and cost-effective manner. Finally,they might be combined with micro-electronic fabrication technology.

One aspect of the invention comprises techniques for treating thesurface of solid-state nanopores in order to either improve theirsequencing performance or to enable the creation of an hybridprotein/solid-state nanopore. In such a hybrid, the solid-state poreacts a substrate with a hole for the protein nanopore, which would bepositioned as a plug within the hole. The protein nanopore would performthe sensing of DNA molecules. This hybrid can the advantages of bothtypes of nanopores: the possibility for batch fabrication, stability,compatibility with micro-electronics, and a population of identicalsensing subunits. Unlike methods where a lipid layer much larger thanthe width of a protein nanopore is used, the hybrid nanopores aregenerally constructed such that the dimensions of the solid state poreare close to the dimensions of the protein nanopore. The solid statepore into which the protein nanopore is disposed is generally from about20% larger to about three times larger than the diameter of the proteinnanopore. In preferred embodiments the solid state pore is sized suchthat only one protein nanopore will associate with the solid state pore.An array of hybrid nanopores is generally constructed by first producingan array of solid state pores in a substrate, selectivelyfunctionalizing the nanopores for attachment of the protein nanopore,then coupling or conjugating the nanopore to the walls of the solidstate pore using liker/spacer chemistry.

FIG. 5 shows an embodiment of the invention illustrating the chemistryused to produce an array of hybrid nanopores of the invention. The solidstate pore can be constructed of one or multiple materials. In FIG. 5,two materials, S1 and S2 are used. In other cases, a single material canbe used. Where two materials are used, for example, both the top and thebottom S1 layers can be fabricated using Al/AlOx, and S2 can comprise agold layer. S2 can be used as a secondary material to facilitatecontrolled surface modification for attachment of the protein nanopore.This control would allow for more precise control over the position ofan attached protein inside a nanopore. In one embodiment, phosphonatepassivation chemistry specific towards S1-Aluminum is used, and thiolchemistry, specific to the gold portion of the sidewall, S2 is used. Thethiol groups functionalizing S2 comprise pendant groups that attach tothe linker/spacer which can be, for example, a protein or otherbiological molecule disposed at a controlled distance from the solidstate pore sidewall and bottom/top. The size of the linker spacermolecule can be tailored to provide the appropriate spacing, for exampleby controlling molecular weight. By using organic molecules such asproteins, the spacers have enough flexibility to accommodate thedifferent spacings which can result, for example from manufacturingvariances in the size of the solid state pore. This control can beuseful for controlling reagent diffusion in/out of the hybrid nanoporesas well as spacing the protein to eliminate conformational restrictionsand to potentially maximize signal to noise within a finite observationvolume. The parameters can be controlled by adjusting the dimensionslabeled as a, b, c, d, and e on the schematic illustration.

One aspect of the invention comprises devices and methods for obtaininga solid state pore sequencing device having a high portion of poreshaving only one nanopore per solid state pore. Protein nanoporesembedded in a lipid membrane can suffer from the issue ofPoisson-loading (loading of a single protein nanopore in each lipidmembrane follows Poission statistics), in this case only a singleprotein nanopore will fit into each solid-state nanopore. With thepresent invention, the pores can be made and functionalized such thatone nanopore is generally present in one solid state pore.

One aspect of the invention comprises the use of surface monolayers on asolid state pore. In some embodiments, SiN substrates are treated usingfunctional methoxy-, ethoxy-, or chloro-organosilane(s) such as —NHSterminated, —NH2 (amine) terminated, carboxylic acid terminated, epoxyterminated, maleimide terminated, isothiocyanate terminated, thiocyanateterminated, thiol terminated, meth(acrylate) terminated, azide, orbiotin terminated. These functional groups for the non-specificimmobilization of aHL or another protein. In some cases, S1 isfunctionalized to have only passive, inactive functional groups on theS1 surface. These functional groups can include polymeric chains atcontrolled length to prevent non-specific adsorption of biologicalspecies and reagents across the S1 surface. Some examples of thesefunctional groups are PEG, fluorinated polymers, and other polymericmoieties at various molecular weights. This chemistry is schematicallyillustrated as (X) and typically provides a passive layer to preventnon-specific noise throughout the detection signal of the hybridnanopore.

In some embodiments, SiOx substrates are treated using functionalorganosilane(s) such as —NHS terminated, —NH2 (amine) terminated,carboxylic acid terminated, epoxy terminated, maleimide terminated,isothiocyanate terminated, thiocyanate terminated, thiol terminated,meth(acrylate) terminated, azide, or biotin terminated. These functionalgroups are useful for non-specific immobilization of aHL or anotherprotein. For specific control over location and conformation of suchproteins inside a hybrid nanopore, S1 can be functionalized to have onlypassive, inactive functional groups on the S1 surface. These functionalgroups may include polymeric chains at controlled length to preventnon-specific adsorption of biological species and reagents across the S1surface. Some examples of these functional groups are PEG, fluorinatedpolymers, and other polymeric moieties at various molecular weights.This chemistry is schematically illustrated as (X) and typicallyprovides a passive layer to prevent non-specific noise throughout thedetection signal of the hybrid nanopore.

In some embodiments, ALD alumina (as substrate) is modified usingphophonate chemistry. This includes phosphate, sulfonate, and silanechemistries since they all have weak affinities towards AlOx surfaces aswell. The phosphonates can have any of the above chemistries on theterminus for surface treatment.

Where gold is the substrate, the invention comprises the use offunctionalized thiol chemistries. The S2 layer is positioned to controlthe depth as which the protein or biological of choice is immobilizedwithin the hybrid nanopore. The distance e in the figure controls thespacing of the linker/spacer such as a protein within the hybridnanopore. The size of the liker/spacer can be adjusted by selecting theappropriate polymeric or rigid chemical spacer length of the linkerbetween S2 and the protein attachment point. For example, this parametercan be controlled via the molecular weight and rigidity of the polymericor non-polymeric linker chemistry used. Also, this can be controlled bythe S2 electrode protrusion into hybrid nanopore. The linker chemistryused to attach alpha-HL or another protein to the hybrid nanoporesidewall substrate can consist of the pendant groups mentioned above,but may or may not also include a polymeric or rigid linker that furtherpositions the protein into the center of the nanopore. This linker candistance can be controlled via control over the molecular weight andchemical composition of this linker. Some examples can includepolypeptide linkers as well as PEG linkers.

The chemistries described above can be used as a conjugation mechanismfor attachment of large molecule sensors such as proteins or quantumdots or functionalized viral templates or carbon nanotubes or DNA, ifthe nanopore is 10s-100s of nanometers in diameter. These large moleculesensors can be used to optically or electrochemically enhance detectionvia molecule-DNA interactions between H-bonds, charge, and in the caseof optical detection via a FRET, quenching, or fluorescence detectionevent.

For example, if the nanopores are ˜1 nm to 3 nm in diameter, the acidterminated silanes can be used to functionalize pores for better controlover DNA translocation. Further, PEGylation with short PEGs may allowfor passivation of pores to allow for ease of translocation.

In some embodiments, the invention provides surface chemistries for theattachment of proteins such as alpha-hemolysin to the solid state poresurface. Functional surface chemistries described above can be used toeither A) conjugate protein via an engineered or available peptideresidue to the nanopore surface, to anchor the protein or B) tofunctionalize the surface chemistry such that the hydrophilic region ofthat chemistry is presented to the surface to facilitate lipid bi-layersupport. White et al., J. Am. Chem. Soc., 2007, 129 (38), 11766-11775,show this using cyano-functionalized surfaces, but any hydrophilicsurface chemistry such as cyano-, amino-, or PEG terminated chemistriesshould support this function.

Specifically, the covalent conjugation of alpha hemolysin (or otherproteins) to the surface of a solid state pore can be achieved viacystine or lysine residues in the protein structure. Further conjugationcould be achieved via engineered peptide sequences in the proteinstructure or through CLIP or SNAP (Covalys) chemistries that arespecific to one and only one residue engineered onto the proteinstructure. In more detail, protein lysine residues can be conjugated toNETS-containing chemistries, cystine residues to maleimide containingsurface chemistries or SNAP to benzyl guanine/SNAP tags introduced ontothe protein and CLIP to benzyl cytosine tags introduced onto the proteinof choice.

One aspect of the invention comprises controlled and un-controlledpolymerization approaches on pores. The synthesis of silane chemistriesthat involve silane monolayers consisting of aphotocleavable/photoinitiatable group that can be used to graft polymersfrom the surfaces of nanopores is known. One example is from thisliterature is N,N(diethylamino)dithiocarbamoylbenzyl(trimethoxy)silane.While this work has been primarily conducted on derivatized SiOxsurfaces (Metters et al) or derivatized polymeric surfaces(Anseth/Bowman et al), polymeric chains can potentially be grown fromthe sidewalls of nanopores to control diameter, functionality, DNAtranslocation speed, and passivation for optical and/or electrochemicaldetection platforms. The initiation kinetics can be slowed down using achain transfer or radical termination agent such as a tetraethylthiuramdisulfide or a thiol, to achieve potential for more precise chainlengths on the functionalized nanopore.

Uncontrollable grafting of polymers to the surface of nanopores could beachieved via polymerization of functional chains (in solution) that canbe attached via conjugation through any of the silanes listed in above.This achieves the same functional nanopore via a “grafting to” approachinstead of a “grafting from” approach.

The polymerization techniques described above can also be used tosupport lipid bi-layer formation for protein immobilization support orfor direct covalent attachment of proteins to surfaces as discussed inIb1-2. The interesting facet of grafting polymer chains to or from thesurface of a nanopore is the ability to control pore diameter, function,mobility (diffusion of molecules through), by controlling molecularweight, density, length, or multifunctionality of these chains. Thisoffers a more fine-tuned way to control bi-layer formation for aHL ormethods for covalently attaching proteins with polymeric chains that canspace the protein from side-walls of the nanopore substrate.

If using a polymeric approach described above, poly(acrylic acid) PAA oradditional charged polymeric chemistries like NIPAAM or other hydrogelscan be used to functionalize nanopores to create an electro-osmotic flowvalve that changes inner-diameter based off pH or directionality viacharge potential. This approach can be useful for governing the rate atwhich DNA translocated through a modified solid state pore and also toreanalyze DNA multiple times.

The devices of this invention can use H-bond interactions betweenfunctionalized electrodes with phosphate groups on ssDNA passing throughthe nanopore as described by Lindsay et al.

As described above, the hybrid nanopores of the present invention aregenerally prepared such that only a single protein nanopore willassociate with each solid state pore by appropriately sizing the solidstate pore and by using linker/spacer chemistry of the appropriatedimensions. In some cases, the solid state pores can accommodate morethan one protein nanopore, and other approaches are used to ensure thatonly one protein nanopore is loaded into one pore, hole, or aperture inthe device. Both the hybrid nanopores described above and the othernanopores used herein can include the use of a lipid layer forsupporting the protein nanopore and acting as a spacer within the solidstate pore.

In some cases loading can be done at a concentration at which a Poissondistribution dictates that at most about 37% of the apertures will havea single nanopore. Measurements on the pores will reveal which of thepores in the array have a single protein nanopore, and only those areused for sequencing measurements. In some cases loadings of singleprotein nanopores higher than that obtained through Poisson statisticsare desired.

In some cases, repeated loading at relatively low concentrations can beused in order improve fraction of single protein nanopores. Where eachof the pores can be addressed independently with a drive voltage, eachpore could be connected to a fluidic conduit that supplies proteinnanopores at a low concentration to the solid state pores, where theeach conduit has a valve which can be controlled to allow or shut of theflow of fluid. The current across the solid state pore is monitoredwhile the flow of fluid is enabled. Measurement of current while loadinga lipid bilayer has been shown, see, e.g. JACS, 127:6502-6503 (2005) andJACS 129:4701-4705 (2007). When a protein nanopore becomes associatedwith the nanopore, a characteristic current/voltage relationship willindicate that a single pore is in place. At the point that a proteinnanopore is associated, the flow of the liquid is interrupted to preventfurther protein nanopore additions. The system can additionally beconstructed to apply an electrical pulse that will dislodge the proteinnanopore from the solid state pore where the electronics indicates thatmore than one protein nanopore has been incorporated. Once the multipleprotein nanopores are removed, the flow of protein nanopores to thesolid state pore can be resumed until a single protein nanopore isdetected. These systems can be automated using feedback to allow theconcurrent loading of multiple wells in the array without active userintervention during the process.

In some cases, steric hindrance can be used to ensure that a singleprotein nanopore is loaded into a single solid state pore. For exampleeach protein nanopore can be attached to a sizing moiety that the sizeof the protein nanopore and the sizing moiety is such that only one willfit into each solid state pore. The sizing moiety can comprise, forexample, one or more of a bead, nanoparticle, dendrimers, polymer, orDNA molecule whose size is on the order of the region between theprotein nanopore and the solid state pore. These methods can be used incombination with membranes such as lipid bilayers. In some cases, thesizing moieties are removed after loading and before measurement.Alternatively, in some cases, the sizing moieties can remain associatedwith the protein nanopores after loading. In some embodiments, multiplesizing moieties are employed. Where membranes such as lipid bilayers areemployed, each protein nanopore can be functionalized with arms, e.g.dendrimers-like arms, each having a membrane inserting moiety at its end(for example a non-porous transmembrane protein). The membrane insertingmoieties will prevent the association of a second protein nanoporecomplex from entering the bilayer.

Electrostatic repulsion can also be used in order to obtain singleprotein nanopore loadings. Each polymer nanopore can be attached to abead, nanoparticle, dendrimers, polymer, or DNA molecule that is highlycharged. The charged protein nanopore complex in the pore will repelother charged protein nanopore complexes. In some cases, the chargedmoieties are removed after loading and before measurement.Alternatively, in some cases, the charged moieties can remain associatedwith the protein nanopores after loading. Charged protein-nanoporecomplexes can also be used with the systems in which attachment of theprotein nanopore into the pore is actively monitored. The charged moietycan be used to actively remove the protein nanopore from the solid statepore using an electric field.

Optical trapping can also be employed in order to obtain single proteinnanopore loadings. Optical traps can be used to capture complexescomprising a bead and a single nanopore protein. The bead can then bepositioned over the solid state pore and released. Multiple pores can beloaded by sequential loading using a single optical trap, or an array ofoptical traps can be used to load multiple pores concurrently. The beadsize and the laser power of the optical trap can be chosen such that nomore than one bead at a time can be captured in the optical trap. Afterloading the protein nanopore into the solid state pore, the bead can becleaved and washed away.

The protein nanopore to be inserted can be wild type or geneticallyengineered. The protein nanopore can comprise a fusion protein with anexonuclease or can be chemically linked to an exonuclease for sequencingusing an exonuclease as described herein. Where an exonuclease isattached, it may have a DNA molecule, such as a template DNA bound to itat the time of loading. This DNA molecule can act as a moiety to providesteric or electrostatic hindrance as described above.

III. Methods of Fabricating Nanopore Sequencing Devices

One aspect of the invention involves the integration of nanoporemicrofluidics with CMOS technology. The integration of thesetechnologies can be important obtaining the cost and reproducibilityrequired for mass-production of a parallelized electronic nanoporesequencing system.

One aspect of the invention is a method of fabricating a multiplexpolymer sequencing device having microfluidic and electronic featuresfrom a semiconductor substrate comprising: obtaining a semiconductorsubstrate; processing the semiconductor substrate to create an array ofmicrofluidic features, wherein the microfluidic features are capable ofsupporting nanopores; and subsequently creating circuit elements on thesubstrate that are electronically coupled to the microfluidic features.In some cases the circuit elements are CMOS circuit elements. In somecases the CMOS circuit elements comprise amplifiers, analog to digitalconverters.

We have found that in fabricating a nanopore polymer sequencing devicefrom a semiconductor substrate in which the semiconductor substratecomprises both microfluidic and electronic features. In such cases, wehave found that in some cases there are advantages to first creating anarray of microfluidic features, and only subsequently adding theelectronic features, for example by CMOS processing. One advantage ofthis approach is that the electronic features are not subjected to theconditions required for creating the microfluidic features, includinghigh temperatures and harsh chemical agents. Processing steps, such asplanarization can be employed after creating the microfluidic featuresand before producing the electronic features.

One aspect of the invention is a method of fabricating a polymersequencing device comprising the following steps in the order presented:obtaining a semiconductor substrate; processing the semiconductorsubstrate to create an array of CMOS circuits, without carrying out analuminum deposition step; processing the semiconductor substrate havingthe CMOS circuits to produce microfluidic features, wherein themicrofluidic features are capable of supporting nanopores; andsubsequently performing an aluminum deposition step to create conductivefeatures. In some cases the processing of step (c) to create themicrofluidic features subjects the semiconductor substrate totemperatures greater than about 250° C.

We have found that in fabricating a nanopore polymer sequencing deviceform a semiconductor substrate having both microfluidic and electronicelements, that in some cases it is advantageous to prepare theelectronic elements, for example, by CMOS, and subsequently preparemicrofluidic features. We have found, however, that where this is done,any processes involving the introduction of aluminum should generallynot be performed until after the creation of the microfluidic features.This approach has the advantage that the final device has aluminumfeatures that may be advantageous for sensitive electronic measurements,but that the aluminum is introduced after the fabrication of themicrofluidic features on the substrate. This process is advantageous inthat aluminum features can be damaged above about 200 or 250, limitingthe ability to effectively create microfluidic features without damagingthe aluminum features.

The integration of an array of electrical/CMOS components (amplifiers)and bio/fluidics components (membranes/solutions/enzymes etc) can beachieved as described herein with a flip-chip technology approach. Inthis approach component layers are processed separately throughout someor all of their production processes, and are matched at or near the endof the assembly process. The separate process flows can be optimizedindependent of each other. In some embodiments, the process allows forthe CMOS layer to be outsourced to a semiconductor foundry where, forexample, only standard processes are required.

In one embodiment, the nanopore/electrode is produced with aself-aligned etching process. A schematic for one embodiment of thisprocess is shown in FIG. 6. The process can start with an insulatorlayer such as a glass wafer. Channels and/or other microfluidic featuresare etched into the glass, for example with a highly directional dryetch process. As shown in FIG. 6, step (I), this insulator substrate canthen be bonded with a wafer bond process a wafer (e.g. silicon wafer).This wafer can be used, for example to pattern electrodes.

As shown in step (II) a selective wet etch process can be used to createa self-aligned array of cavities, or discrete regions, in the siliconwafer. If necessary, the Si wafer can be thinned as shown in step (III)to remove excess material. As shown in steps (III) and (IV), individualelectrodes can be defined by patterning the Si wafer withphotolithography and a dry etch. An advantage of this self-alignedetching process, is that the alignment of the etch mask and the glassholes/cavities can be done without highly accurate alignment processes.Metal pads can be evaporated on each electrode to provide betterelectrical contact. This can be done before or after the electrode etchstep. The process can be used to create an individually containedelectrode for each measurement site.

One aspect of the invention is a method for fabricating a polymersequencing device comprising: producing an insulator layer havingmicrofluidic elements comprising an array of pores extending through theinsulator; bonding the insulator layer with a semiconductor layer;exposing the semiconducting layer to etchant through the pores in theinsulator to produce discrete reservoirs in the semiconductor layer;removing portions of the semiconductor layer to isolate the discretereservoirs, and providing electrical contacts that allow current to bedirected to each of the discrete reservoirs; bonding an electric circuitlayer to the semiconducting layer such that the electric circuits on theelectric circuit layer are electrically connected to the electricalcontacts on the semiconductor layer.

In some cases the method further comprising the step of adding nanoporesinto each of the pores. The nanopores can comprise solid statenanopores, protein nanopores, or hybrid solid state/protein nanopores.In some cases the method comprises the use of two or more electrodeswithin the discrete reservoir.

One aspect of the invention is a method for fabricating a polymersequencing device comprising: producing an insulator layer havingmicrofluidic elements comprising an array of pores extending through theinsulator; bonding the insulator layer with a semiconductor layerwherein the semiconducting layer comprises an array of wellscorresponding to the pores on the insulator layer, whereby the bondingproduces an array of discrete reservoirs; removing portions of thesemiconductor layer to isolate the discrete reservoirs, and providingelectrical contacts that allow current to be directed to each of thediscrete reservoirs; and bonding an electric circuit layer to thesemiconducting layer such that the electric circuits on the electriccircuit layer are electrically connected to the electrical contacts onthe semiconductor layer.

An alternative embodiment involves starting to with a Si wafer, growinga thick field oxide on top of the wafer, and patterning the oxide as wasdone above for the insulator layer. The subsequent steps described abovecan be used to produce a nanopore array.

In some embodiments, the signals coming out of the electrodes will beamplified in a CMOS amplifier stage. Each electrode can be matched upwith its own amplifier stage by using flip chip technology as shown instep (V) of FIG. 6. In this approach a CMOS amplifier array is patternedon a Si wafer, with pitch and dimensions matching the electrode array onthe bio component. The top of the CMOS chip consists of a matching arrayof electrodes (metal I/O pads). The input/output pads on the amplifierchip are bonded to the matching electrodes of the bio chip assembly.This can be done with solder bumps, thermally or ultrasonically.

In some embodiments microfluidic features can be created in thesemiconductor substrate prior to wafer bonding. FIG. 7 shows thecreation of microfluidic features. In step (I) an array of wells iscreated in a semiconductor substrate. In step (II), an insulator layerhaving microfluidic elements and pores extending through the insulatinglayer is wafer bonded with the semiconductor substrate such that thearray of pores aligns with the array of wells to produce an array ofcavities. In some embodiments, circuits are created on the semiconductorsubstrate as described above, for example using CMOS processes.

In some aspects of the invention, a SOI wafer is used as the substratefor creating the nanopore sequencing device. Fore example, with an SOIsubstrate having a top silicon layer, an insulator (oxide) layer, and abottom silicon layer, the top silicon can be used as a top electrode, ora top electrode can be built onto the top electrode. The intermediaryoxide layer can be used as the layer which contains the nanometer scaleaperture, such as a nanopore protein within a supporting lipid bi-layer.In some embodiments, the bottom silicon can serve as serve as a ground.Once the SOI based device is constructed, polymeric materials such aspolydimethylsiloxane can be used to produces microfluidic features suchas channels and reservoirs. For example, in some cases, the device couldbe sealed with simple PDMS chips.

In some embodiments, electronic circuits and electrodes can be builtinto top and/or the bottom silicon layer, and the circuits can beelectrically coupled to the fluidic regions surrounding the nanopore. Inone embodiment, the top silicon in the SOI wafer is used to build anop-amp, which can be used to boost the signal prior to measuring thecurrent. In some cases, full CMOS circuitry can be incorporated. In somecases, less complex circuitry can be incorporated, for example with theinclusion of a simple op-amp. The op-amp could provide some a benefit ofnoise immunity. The electric circuits on the chip, for example, theop-amp would generally be electrically isolated from the fluid, eitherthrough a dielectric coating (Si3N4, SiO2) or by a PDMS chip.

FIG. 8 shows a schematic for a process using an SOI wafer. In step (I),portions of the top silicon layer and the bottom silicon layer areremoved to expose regions of the insulator (oxide) layer. This processcan produce, for example, an array of regions in which the insulator isexposed on both sides. Step (I) also comprises the addition of circuitsand electrodes into the top silicon layer. In some embodiments,electrodes and/or circuits can also be added to the bottom layer. Instep (II) of FIG. 8, a pore is created in the insulator. This pore canbe used to hold the nanopore of the invention which can be fabricatedinto the pore, or added to the pore subsequently as known in the art andas described herein.

FIG. 9 illustrates that polymers such as PDMS can be used to fluidicallyseal portions of the device. In some cases, as shown in FIG. 9,electrical connections can be provided to electrodes on the devicethought the polymer layers.

In some embodiments, the devices of the invention are built having acommon ground design. Having a common ground avoids the complexityassociated with providing separate pairs of electrodes for each well. Insome cases, the bottom of each of the cells is electrically connected toprovide a common ground. The ground produced in this manner could befloated to the best potential for the experiment. For example, as thereaction progresses, and species are generated, the potential of thesolution may change.

In some aspects of the invention a structure which provides 4-pointprobing is created. 4-point probes are well known in the art to providefor accurate electrical measurements. The 4-point probe designs of theinvention can be produced on glass wafers with electrodes such as gold(Au) or platinum (Pt) electrodes. They can also be produced on SOI orSOI-like wafers. In the 4 point probe designs of the invention, twolarge electrodes provide the drive current, and two smaller electrodesare used to measure potential drop across the bi-layer. As describedherein, in some embodiment, the 4-point measurements of the inventioninvolve using drive electrodes which drive the current through multiplenanopores, while having pairs of measurement electrodes for each of thenanopores. The smaller electrodes can be connected to a high impedancecircuit to get good quality measurement characteristics while the driveelectrodes are connected to a stable power supply.

One aspect of the invention is a method for fabricating a polymersequencing device comprising: obtaining an SOI substrate comprisinghaving a top silicon layer, an insulator layer, and a bottom siliconlayer; processing the top silicon layer and bottom silicon layer toremove portions of each layer to produce an array of exposed regions inwhich both the top and bottom surfaces of the insulator layer areexposed; processing the top silicon layer or the bottom silicon layer orboth the top silicon layer and bottom silicon layer to add electrodesand electrical circuits; and processing the insulator layer to producean array of pores through the exposed regions of the insulator layer.

In some cases the method further comprises adding polymer layers toproduce microfluidic features. In some cases the method furthercomprises inserting a nanopore into the pores in the insulator layer.

Where a protein nanopore such as alpha hemolysin is used as thenanopore, the nanopores can be fabricated by, for example, coating aportion of a pore within the device with a primer to which the lipidlayer or other supporting linker/spacer will associate. In some cases,the level of a solution that is in contact with the holes into which thepores are to be deposited can be raised or lowered such that the surfaceof the liquid is disposed within the hole at the desired level. Surfaceactive agents on the liquid can then react with the nanopore at thelevel at which the surface of the liquid contacts the pore. This cancreate a functionalized region of the hole that can be used tospecifically interact with the lipid layer or linker/spacer.

IV Nanopore Sequencing Systems

The invention includes sequencing system which incorporate the devicesand methods described herein. The systems of the invention incorporatethe multiplex nanopore polymer sequencing device described herein, andalso include a processing system for driving the electronics, and aprocessing system for gathering, storing, and analyzing the dataproduced.

Generally, the raw data from the sequencing run will be processed byvarious algorithms in order to correlate the electronic measurementswith the sequence of the polymer. Some algorithms that can be used toincrease the base calling capability of the devices are describedherein, others are known in the art. In some cases, the systems of theinvention incorporate feedback capability, allowing for changing thesequencing conditions dynamically due to measured signals. Somealgorithms for dynamic measurements are described herein. The systems ofthe invention will also provide for handling and introducing samplesinto the devices.

V. Methods of Nanopore Sequencing

The invention comprises methods of sequencing using the multiplexpolymer sequencing devices described herein.

Enzymatic Control of Translocation Rate

One aspect of the invention comprises controlling the translocation of apolymer molecule through the nanopore. For the purposes of singlemolecule sequencing it can be advantageous to control the translocationof DNA through nanopore structures under applied voltage. See, forexample US Patent Application 2006/0063171. Protein components on eitherthe cis or trans side of the nanopore can be utilized to control therate of the translocation through the nanopore, which can facilitatecertain sequence detection methods. Shown in diagrammatic form in FIG.10 is the passage of DNA or RNA (101) translocating under an appliedvoltage though a nanopore structure (102) within a physical barrier(103). Proteinaceous components can be located on either or both sidesof the nanopore structure (100, 104) to interact with the translocatingnucleic acid strands. Optionally, one or more of the interactingcomponents can be covalently, or non covalently tethered to the nanoporestructure (102) or barrier (103) as indicated below.

The proteins can be chosen from a host of DNA or RNA metabolizing ortranslocating enzymes (see, e.g., FIG. 10), or DNA or RNA bindingproteins (see, e.g., FIG. 11). For example, these enzymes can be chosenfrom various polymerases including, but not limited to, phi29 DNApolymerase, T7 DNA pol, T4 DNA pol, E. coli DNA pol 1, Klenow fragment,T7 RNA polymerase, and E coli RNA polymerase, as well as associatedsubunits and cofactors. The nucleic acid strand translocating throughthe nanopore can be comprised of either the template or a nascent strandsynthesized by the polymerase, e.g., a displaced nascent strand (e.g.,from a rolling circle amplification reaction) or an RNA transcript.Optionally, the protein components can be chosen from a broad class ofDNA translocation enzymes including DNA and RNA helicases, viral genomepackaging motors, and chromatin remodeling ATPases. Certain examples ofsuch protein components are described, e.g., in: Mechanisms fornucleosome movement by ATP-dependent chromatin remodeling complexes.Saha A, Wittmeyer J, Cairns B R. Results Probl Cell Differ. 2006;41:127-48, Mechanisms of nucleic acid translocases: lessons fromstructural biology and single-molecule biophysics. Hopfner K P,Michaelis J. Curr Opin Struct Biol. 2007 February; 17(1):87-95. Epub2006, Structure and mechanism of helicases and nucleic acidtranslocases. Singleton M R, Dillingham M S, Wigley D B. Annu RevBiochem. 2007; 76:23-50, Non-hexameric DNA helicases and translocases:mechanisms and regulation. Lohman T M, Tomko E J, Wu C G. Nat Rev MolCell Biol. 2008 May; 9(5):391-401.

In a preferred mode of operation, the rate of nucleic acid translocationcan be controlled by the concentration of a reactant or cofactor. Forexample, DNA translocases couple hydrolysis of nucleotide triphosphatecofactors to the translocation of DNA. The E. coli FtsK enzyme canadvance the DNA at speed of about 5000 bases per second (at 25° C.) byhydrolyzing ATP. Under conditions of limiting ATP the rate can bemodulated to slow the translocation rate for optimal sequence detection.FtsK enzyme can translocate DNA in either direction which can beutilized in such a configuration to facilitate redundant single moleculesequencing to increase consensus accuracy. It is understood by thoseskilled in the art that similar modes of control of DNA translocation bypolymerases and helicases could likewise be affected by theconcentration of nucleotide or metal cofactors. Redundant sequencingapproaches could also be affected by intrinsic or extrinsic exonucleaseactivities. (See, e.g., U.S. Pat. No. 7,476,503; and U.S. Ser. No.12/413,258, filed Mar. 27, 2009, both of which are incorporated hereinby reference in their entireties for all purposes.) The kinetics of theenzymes can be altered by mutation or conditions to maximize thelikelihood of sequence detection. (See, e.g., U.S. Ser. No. 12/414,191,filed Mar. 30, 2009; and U.S. Ser. No. 12/384,112, filed Mar. 30, 2009,both of which are incorporated herein by reference in their entiretiesfor all purposes.)

The present invention is generally directed to improved enzyme reactioncompositions, methods, and systems that exhibit kinetic mechanismshaving two or more slow, kinetically observable, or partiallyrate-limiting reaction steps within an observable phase of thepolymerase reaction. Such systems can be useful for example, insingle-molecule, real-time observations of such enzyme activity, whichrely, at least in part, on detecting and identifying the enzyme reactionas it is occurring. By designing the reaction system to have two or morepartially rate-limiting steps, the relative number of short, difficultto detect, events can be lowered. Enzymatic reactions often occur atrates that can far exceed the speed of a variety of detection systems,e.g., optical detectors. As such, by providing two or more partiallyrate-limiting steps within a phase of an enzyme reaction, one improvesthe ability to monitor that reaction using optical detection systems.

One particular exemplary system includes compositions for carrying outsingle-molecule DNA sequencing. We describe systems that exhibit twoslow steps within an observable phase. An observable phase willgenerally have a time period during which the phase is observable. Thetime period for a bright phase, for example, can be represented by thepulse width. The time period for a dark phase can be represented, forexample, by the interpulse distance. The length of each time period willnot be the same for each nucleotide addition, resulting in adistribution of the length of the time periods. In some cases, the timeperiods with the shortest length will not be detected, leading toerrors, for example in single-molecule sequencing. We have found that bydesigning enzyme systems such as polymerase reaction systems in whichthere are two slow, or kinetically observable, steps within anobservable phase, the relative number of short, unobservable, timeperiods can be reduced, resulting in a higher proportion of observablesequencing events, and allowing for a more accurate determination ofnucleotide sequence. As used herein, an observable phase includes phasesthat are not directly observable, but can be ascertained by measurementsof other, related phases. For example, the lengths of dark phases can beobserved by measuring the times between optical pulses corresponding toa related bright optical phase. Also as described herein, a phase whichis dark under some labeling conditions can be bright under otherlabeling conditions.

While primarily described in terms of nucleic acid polymerases, andparticularly DNA polymerases, it will be appreciated that the approachof providing multiple slow, or kinetically observable steps, within anenzyme system is applicable to other enzyme systems where one may wishto directly observe the enzyme reaction, in real time. Such enzymesystems include, for example, other synthesizing enzymes, e.g., RNApolymerases, reverse transcriptases, ribosomal polymerases, as well asother enzyme systems, such as kinases, phosphatases, proteases,nucleases, ligases, and the like.

Polymerase-Mediated Synthesis

In natural polymerase-mediated nucleic acid synthesis, a complex isformed between a polymerase enzyme, a template nucleic acid sequence,and a priming sequence that serves as the point of initiation of thesynthetic process. During synthesis, the polymerase samples nucleotidemonomers from the reaction mix to determine their complementarity to thenext base in the template sequence. When the sampled base iscomplementary to the next base, it is incorporated into the growingnascent strand. This process continues along the length of the templatesequence to effectively duplicate that template. Although described in asimplified schematic fashion, the actual biochemical process ofincorporation is relatively complex.

The process can be described as a sequence of steps, wherein each stepcan be characterized as having a particular forward and reverse reactionrate that can be represented by a rate constant. One representation ofthe incorporation biochemistry is provided in FIG. 32. It is to beunderstood that the scheme shown in FIG. 32 does not provide a uniquerepresentation of the process. In some cases, the process can bedescribed using fewer steps. For example, the process is sometimesrepresented without inclusion of the enzyme isomerization steps 106 and110. Alternatively, the process can be represented by includingadditional steps such as cofactor binding. Generally, steps which can beslow, and thus limit the rate of reaction will tend to be included. Thepresent invention relates to methods, systems, and compositions in whichthe polymerization reaction has two or more slow steps within certainphases of the polymerase reaction. Various schemes can be used torepresent a reaction having two slow steps that may have more or feweridentified steps. In some cases the two or more slow steps areconsecutive. In some cases, there can be intervening fast steps betweenthe two or more slow steps.

As shown in FIG. 32, the synthesis process begins with the binding ofthe primed nucleic acid template (D) to the polymerase (P) at step 102.Nucleotide (N) binding with the complex occurs at step 104. Step 106represents the isomerization of the polymerase from the open to closedconfiguration. Step 108 is the chemistry step where the nucleotide isincorporated into the growing strand of the nucleic acid beingsynthesized. At step 110, polymerase isomerization occurs from theclosed to the open position. The polyphosphate component that is cleavedupon incorporation is released from the complex at step 112. Thepolymerase then translocates on the template at step 114. As shown, thevarious steps can include reversible paths and may be characterized bythe reaction constants shown in FIG. 32 where:

k_(on)/k_(off)=DNA binding/release;

k1/k−1=nucleotide binding/release;

k2/k−2=polymerase isomerization (open/closed);

k3/k−3=nucleotide incorporation (chemistry);

k4/k−4=polymerase isomerization (closed/open);

k5/k−5=polyphosphate release/binding;

k6/k−6=polymerase translocation.

Thus, during steps 104 through 110, the nucleotide is retained withinthe overall complex, and during steps 104 and 106, reversal of thereaction step will yield an unproductive event, i.e., not resulting inincorporation. For example, a bound nucleotide at step 104 may bereleased regardless of whether it is the correct nucleotide forincorporation.

By selecting the appropriate polymerase enzyme, polymerase reactionconditions, and polymerase substrates, the absolute and relative ratesof the various steps can be controlled. We have found that controllingthe reaction such that the reaction exhibits two or more kineticallyobservable, or slow steps can produce a nucleic acid polymerizationreaction in which the incorporation of the nucleotides can be observedmore accurately. These characteristics are particularly useful forsequencing applications, and in particular single-molecule DNAsequencing.

In some cases, the invention involves a process having two or morekinetically observable steps that comprise steps after nucleotidebinding through the step of product release. For the mechanism shown inFIG. 32, this would be, for example, any of steps 106, 108, 110, and112. In some cases, steps 108 (nucleotide incorporation) and 112(product release) are the two slow, or kinetically observable steps. Asnoted previously, where one desires systems with slow steps in a darkphase, the invention may involve a process having two or more slow stepsthat comprise the steps after product release through nucleotidebinding. For the mechanism shown in FIG. 32, this would include steps114 and 104.

By the term slow-step we generally mean a kinetically observable step orpartially rat-limiting step. The slow step need not be slow in theabsolute sense, but will be relatively slow as compared with other stepsin the enzymatic reaction. The slow, or kinetically observable steps,can be, for example, each partially rate-limiting, in that the rate ofthe step has a measurable effect on the kinetics of the enzymaticreaction. An enzymatic process, such as nucleic acid polymerization, canhave both slower, kinetically observable steps and faster steps whichcan be so fast that they have no measurable effect on the kinetics, orrate, of the reaction. In some reactions, there can be a singlerate-limiting step. For such reactions, the kinetics can becharacterized by the rate of that single step. Other reactions will nothave a single rate-limiting step, but will have two or more steps whichare close enough in rate such that the characteristics of each willcontribute to the kinetics of the reaction. A kinetically observablestep is generally a step which is slow enough relative to the othersteps in the reaction such that it can be experimentally ascertained.The experimental identification of a kinetically observable step can bedone by the methods described herein, or by methods for assessing thekinetics of chemical and enzymatic reactions known in the art. For thecurrent invention, the slow, or kinetically observable steps, need notbe the slowest step or the rate-limiting step of the reaction. Forexample, a process of the current invention can involve a reaction inwhich step 104, nucleotide addition is the slowest (rate-limiting) step,while two or more of steps 106, 108, 110, or 112 are each kineticallyobservable.

As used herein, the term rate, as applied to the steps of a reaction canrefer to the average rate of reaction. For example, when observing asingle-molecule reaction, there will generally be variations in therates as each individual nucleotide is added to a growing nucleic acid.In such cases the rate of the reaction can be represented by observing anumber of individual events, and combining the rates, for example, byobtaining an average of the rates.

As used herein, the reference to the rate of a step or rate constant fora step can refer to the forward reaction rate of the polymerasereaction. As is generally understood in the art, reaction steps can becharacterized as having forward and reverse rate constants. For example,for step 108, k3 represents the forward rate constant, and k−3represents the reverse rate constant for the nucleotide incorporation.Some reaction steps, such as step 108, constitute steps which would beexpected to be first order steps. Other steps, such as the forwardreaction of step 104, with rate constant k2, would be expected to besecond order rate constants. For the purposes of the invention, forcomparing the rate or the rate constant of a first order to a secondorder step, the second order rate constant k2 can be treated as apseudo-first order rate constant with the value [N]*k2 where theconcentration of nucleotide [N] is known.

It is generally desirable that the kinetically observable steps of theinvention have rate constants that are lower than about 1000 per second.In some cases, the rate constants are lower than about 500 per second,lower than about 200 per second, lower than about 100 per second, lowerthan about 60 per second, lower than about 50 per second, lower thanabout 30 per second, lower than about 20 per second, lower than about 10per second, lower than about 5 per second, lower than about 2 persecond, or lower than about 1 per second.

In some embodiments the slowest of the two or more kineticallyobservable steps has a rate constant when measured under single-moleculeconditions of between about 500 to about 0.1 per second, about 200 toabout 0.1 per second, about 60 to about 0.5 per second, about 30 persecond to about 2 per second, or about 10 to about 3 per second.

The ratio of the rate constants of each the two or more slow steps isgenerally greater than 1:10, in some cases the ratio of the rateconstants is about 1:5, in some cases the ratio of the rate constants isabout 1:2, in some cases, the ratio of rate constants is about 1:1. Theratio of the rate constants can be between about 1:10 and about 1:1,between about 1:5 and about 1:1, or between about 1:2 and about 1:1.

In some cases it is useful to consider the two slow-step system in termsof rates rather than rate constants. It is generally desirable that thekinetically observable steps of the invention have rates that are lowerthan about 1000 molecules per second when the reactions are carried outunder single-molecule conditions. In some cases, the rates are lowerthan about 500 molecules per second, lower than about 200 molecules persecond, lower than about 100 molecules per second, lower than about 60molecules per second, lower than about 50 molecules per second, lowerthan about 30 molecules per second, lower than about 20 molecules persecond, lower than about 10 molecules per second, lower than about 5molecules per second, lower than about 2 molecules per second, or lowerthan about 1 molecule per second.

In some embodiments the slowest of the two or more kineticallyobservable steps has a rate when measured under single-moleculeconditions of between about 500 to about 0.01 molecules per second,between about 200 to about 0.1 molecules per second, between about 60 toabout 0.5 molecules per second, about 30 molecules per second to about 2molecules per second, or about 10 to about 3 molecules per second.

The ratio of the rates of each the two or more slow steps is generallygreater than 1:10, in some cases the ratio of the rates is about 1:5, insome cases the ratio of the rates is about 1:2, in some cases, the ratioof rates is about 1:1. The ratio can be between about 1:10 and about1:1, between about 1:5 and about 1:1, or between about 1:2 and about1:1.

A two or more slow-step system of the present invention can be obtainedby selecting the correct set of polymerase enzyme, polymerase reactionconditions, and polymerase reaction substrates.

While not being bound by theory, we provide the following theoreticalbasis for obtaining improved single-molecule sequencing results by usinga system having two or more slow steps within an observable phase. Whiledescribed here for nucleic acid polymerization, it will be appreciatedthat the two slow step systems of the invention can also be used forimproved observation of other enzyme systems. A model for the effect oftwo slow steps on the probability density for residence time isdescribed herein. FIG. 33 shows a plot of calculated probability densityfor residence time for cases in which (1) one step is rate-limiting and(2) two equivalent partially rate-limiting (slow) steps are present forthe observable phase in which the nucleotide is associated with theenzyme.

For the case in which one step is rate-limiting, the probabilitydistribution for the binding time can be represented by the singleexponential equation:y=A ₀ e ^(−kt)  Eq. 1

This represents the case in which, for example, incorporation ofnucleotide into the growing nucleic acid (step 108 in FIG. 32) is thesingle slow step.

FIG. 33 illustrates that where one slow-step is present in this phase,there is an exponentially decreasing probability of a given residencetime as the residence time increases, providing a distribution in whichthere is a relatively high probability that the residence time will beshort.

For the case in which there are two slow steps in this phase, forexample where both the incorporation step (step 108 in FIG. 32) and therelease of product (PPi) step (step 112 in FIG. 32) are slow, theprobability density versus residence time can be represented by a doubleexponential equation:y=A ₀ e ^(−k) ¹ ^(t) −B ₀ e ^(−k) ² ^(t)  Eq. 2

FIG. 33 illustrates that for the case in where there are two slow steps,the probability of very fast residence times is relatively low ascompared to the case having one slow step. In addition, the probabilitydistribution for two slow steps exhibits a peak in the plot ofprobability density versus residence time. This type of residence timedistribution can be advantageous for single-molecule sequencing where itis desired to measure a high proportion of binding events and where fastbinding events may be unreliably detected.

Typically, for a given illumination/detection system there will be aminimum detection time below which events, such as binding events, willbe unreliably detected or not detected at all. This minimum detectiontime can be attributed, for example, to the frame acquisition time orframe rate of the optical detector, for example, a CCD camera. Adiscussion of detection times and approaches to detection for thesetypes of systems is provided in U.S. patent application Ser. No.12/351,173 the full disclosures of which are incorporated herein byreference in their entirety for all purposes. FIG. 33 includes a linewhich indicates a point where the residence time equals a minimumdetection time (Tmin). The area under the curve in the region below Tminrepresents the population of short pulses which will not be accuratelydetected for this system. It can be seen from FIG. 33 that the relativeproportion of binding times that fall below Tmin is significantly lowerfor the case in which the reaction exhibits two slow steps as comparedto the case where the reaction exhibits one slow step.

The steps that comprise the two slow steps can include, for example,nucleotide addition, enzymatic isomerization such as to or from a closedstate, cofactor binding or release, product release, incorporation ofnucleic acid into the growing nucleic acid, or translocation.

Polymerase Enzyme

One important aspect of obtaining a two slow-step system of theinvention is selection of the enzyme that is used. The polymerase enzymecan be modified in a manner in which the relative rates of the steps ofthe polymerase reactions are changed such that the enzyme will becapable of showing two slow-step characteristics. Recombinant enzymesuseful in the present invention are described, for example, in copendingU.S. patent application Ser. No. 12,384,112 entitled “Generation ofModified Polymerases for Improved Accuracy in Single-moleculeSequencing”, filed Mar. 30, 2009.

A modified polymerase (e.g., a modified recombinant Φ29-type DNApolymerase for example, a modified recombinant Φ29, B103, GA-1, PZA,Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5,PR722, or L17 polymerase) that exhibits one or more slow stepsoptionally includes a mutation (e.g., an amino acid substitution orinsertion) at one or more of positions 484, 249, 179, 198, 211, 255,259, 360, 363, 365, 370, 372, 378, 381, 383, 387, 389, 393, 433, 478,480, 514, 251, 371, 379, 380, 383, 458, 486, 101, 188, 189, 303, 313,395, 414, 497, 500, 531, 532, 534, 558, 570, 572, 574, 64, 305, 392,402, 422, 496, 529, 538, 555, 575, 254, 390, 372-397, and 507-514, wherenumbering of positions is relative to wild-type Φ29 polymerase. Forexample, relative to wild-type Φ29 a modified recombinant polymerase caninclude at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of: an amino acidsubstitution at position 484; an amino acid substitution at position198; an amino acid substitution at position 381; an amino acidsubstitution at position 387 and an amino acid substitution at position484; an amino acid substitution at position 372, an amino acidsubstitution at position 480, and an amino acid substitution at position484; an amino acid substitution at position 372, an amino acidsubstitution at position 387, and an amino acid substitution at position480; an amino acid substitution at position 372, an amino acidsubstitution at position 387, and an amino acid substitution at position484; an amino acid substitution at position 372, an amino acidsubstitution at position 387, an amino acid substitution at position478, and an amino acid substitution at position 484; A484E; A484Y;N387L; T372Q; T372Y; T372Y and K478Y; K478Y; I370W; F198W; L381A; T368F;A484E, E375Y, K512Y, and T368F; A484Y, E375Y, K512Y, and T368F; N387L,E375Y, K512Y, and T368F; T372Q, E375Y, K512Y, and T368F; T372L, E375Y,K512Y, and T368F; T372Y, K478Y, E375Y, K512Y, and T368F; I370W, E375Y,K512Y, and T368F; F198W, E375Y, K512Y, and T368F; L381A, E375Y, K512Y,and T368F; and E375Y, K512Y, and T368F. A K512F substitution (or K512W,K512L, K512I, K512V, K512H, etc.) is optionally employed, e.g., where aK512Y substitution is listed herein. As another example, the modifiedpolymerase can include an insertion of at least one amino acid (e.g.,1-7 amino acids, e.g., glycine) within residues 372-397 and/or 507-514.For example, a glycine residue can be introduced after residue 374, 375,511, and/or 512 (designated as 374.1G, 375.1G, etc.). In someembodiments the enzyme has one or more of the amino acid substitutionsE375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L, K478Y, I370W,F198W, and L381A.

A list of exemplary mutations and combinations thereof is provided inTable 1, and additional exemplary mutations are described herein.Essentially any of these mutations, or any combination thereof, can beintroduced into a polymerase to produce a modified recombinantpolymerase (e.g., into wild-type Φ29, an exonuclease deficient Φ29-typepolymerase, and/or E375Y/K512Y/T368F Φ29, as just a few examples).

TABLE 1 Mutation Rationale D249E metal coordination A484E metalcoordination D249E/A484E metal coordination A484D metal coordinationA484H metal coordination A484Y metal coordination D249E/A484D metalcoordination D249E/A484H metal coordination D249E/A484Y metalcoordination 374.1G/375.1A dye interaction 374.1Gins/375.1Gins dyeinteraction V514Y dye interaction V514F dye interaction511.1G/K512Y/512.1G dye interaction T372H closed conformation of fingersT372V closed conformation of fingers T372I closed conformation offingers T372F closed conformation of fingers T372Y closed conformationof fingers T372N closed conformation of fingers T372Q closedconformation of fingers T372L closed conformation of fingers T372L/K478Yclosed conformation of fingers T372Y/K478Y closed conformation offingers T372Y/K478L closed conformation of fingers K478Y closedconformation of fingers D365N closed conformation of fingers D365Qclosed conformation of fingers L480H closed conformation of fingersL480F closed conformation of fingers L381A closed conformation of fingerand exo I179A closed conformation of finger and exo I378A closedconformation of finger and exo I179A/L381A closed conformation of fingerand exo I179A/I378A/L381A closed conformation of finger and exoI370A/I378A closed conformation of finger and exoI179A/I370A/I378A/L381A closed conformation of finger and exo I179Wclosed conformation of finger and exo I179H closed conformation offinger and exo F211A closed conformation of finger and exo F211W closedconformation of finger and exo F211H closed conformation of finger andexo F198A closed conformation of finger and exo F198W closedconformation of finger and exo F198H closed conformation of finger andexo P255A closed conformation of finger and exo P255W closedconformation of finger and exo P255H closed conformation of finger andexo Y259A closed conformation of finger and exo Y259W closedconformation of finger and exo Y259H closed conformation of finger andexo F360A closed conformation of finger and exo F360W closedconformation of finger and exo F360H closed conformation of finger andexo F363A closed conformation of finger and exo F363H closedconformation of finger and exo F363W closed conformation of finger andexo I370W closed conformation of finger and exo I370H closedconformation of finger and exo K371A closed conformation of finger andexo K371W closed conformation of finger and exo I378H closedconformation of finger and exo I378W closed conformation of finger andexo L381W closed conformation of finger and exo L381H closedconformation of finger and exo K383N closed conformation of finger andexo K383A closed conformation of finger and exo L389A closedconformation of finger and exo L389W closed conformation of finger andexo L389H closed conformation of finger and exo F393A closedconformation of finger and exo F393W closed conformation of finger andexo F393H closed conformation of finger and exo I433A closedconformation of finger and exo I433W closed conformation of finger andexo I433H closed conformation of finger and exo K383L phosphate backboneinteraction K383H phosphate backbone interaction K383R phosphatebackbone interaction Q380R phosphate backbone interaction Q380Hphosphate backbone interaction Q380K phosphate backbone interactionK371L phosphate backbone interaction K371H phosphate backboneinteraction K371R phosphate backbone interaction K379L phosphatebackbone interaction K379H phosphate backbone interaction K379Rphosphate backbone interaction E486A phosphate backbone interactionE486D phosphate backbone interaction N387L incoming nucleotide base andtranslocation N387F incoming nucleotide base and translocation N387Vincoming nucleotide base and translocation N251H phosphate interactionN251Q phosphate interaction N251D phosphate interaction N251E phosphateinteraction N251K phosphate interaction N251R phosphate interactionA484K phosphate interaction A484R phosphate interaction K383Q phosphateinteraction K383N phosphate interaction K383T phosphate interactionK383S phosphate interaction K383A phosphate interaction I179H/I378Hclosed conformation I179W/I378W closed conformation I179Y/I378Y closedconformation K478L I378Y I370A I179Y N387L/A484E N387L/A484YT372Q/N387L/A484E T372Q/N387L/A484Y T372L/N387L/A484ET372L/N387L/K478Y/A484Y T372Y/N387L/K478Y/A484E T372Y/N387L/K478Y/A484Y

Table 2 presents exemplary Φ29 mutants that can exhibit two slow stepbehavior under appropriate reaction conditions. The first three modifiedpolymerases exhibit the most pronounced two slow step behavior, followedby the next six. As noted, the polymerases are optionallyexonuclease-deficient; for example, they can also include an N62Dsubstitution.

TABLE 2 A484E/E375Y/K512Y/T368F A484Y/E375Y/K512Y/T368FN387L/E375Y/K512Y/T368F T372Q/E375Y/K512Y/T368F T372L/E375Y/K512Y/T368FT372Y/K478Y/E375Y/K512Y/T368F I370W/E375Y/K512Y/T368FF198W/E375Y/K512Y/T368F L381A/E375Y/K512Y/T368F E375Y/K512Y/T368FPolymerase Reaction Conditions

The polymerase reaction conditions can also be important for obtaining atwo slow-step enzyme system. In particular, polymerase reactionconditions include components selected to produce two slow-stepkinetics. The polymerase reaction conditions include the type andconcentration of buffer, the pH of the reaction, the temperature, thetype and concentration of salts, the presence of particular additiveswhich influence the kinetics of the enzyme, and the type, concentration,and relative amounts of various cofactors, including metal cofactors.The term “polymerase reaction conditions” as used herein generallyexcludes the concentration of the polymerase enzyme or the concentrationof the primer-template complex. Thus, two reactions are run undersubstantially the same polymerase reaction conditions where the firstreaction has a small amount of polymerase enzyme, such as a singlepolymerase enzyme, and a small amount of primer template complex, suchas a single primer-template complex associated with a single polymeraseenzyme, and the second reaction has a higher concentration of polymeraseenzyme, for example a concentration of polymerase enzyme of about 0.05μM to 0.5 μM, and about 0.01 μM to about 0.1 μM.

It some embodiments the type and concentration of buffer are chosen inorder to produce a reaction having two slow steps. Enzymatic reactionsare often run in the presence of a buffer, which is used, in part, tocontrol the pH of the reaction mixture. We have found that in some casesthe type of buffer can influence the kinetics of the polymerase reactionin a way that can lead to two slow-step kinetics. For example, in somecases, we have found that the use of TRIS as buffer is useful forobtaining a two slow-step reaction. Buffers suitable for the inventioninclude, for example, TAPS(3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine(N,N-bis(2-hydroxyethyl)glycine), TRIS (tris(hydroxymethyl)methylamine),ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid), Tricine(N-tris(hydroxymethyl)methylglycine), HEPES4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TES(2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), and MES(2-(N-morpholino)ethanesulfonic acid).

The pH of the reaction can influence the kinetics of the polymerasereaction, and can be used as one of the polymerase reaction conditionsto obtain a reaction exhibiting two slow-step kinetics. The pH can beadjusted to a value that produces a two slow-step reaction mechanism.The pH is generally between about 6 and about 9. In some cases, the pHis between about 6.5 and about 8.0. In some cases, the pH is betweenabout 6.5 and 7.5. In some cases, the pH is about 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

The temperature of the reaction can be adjusted in order to obtain areaction exhibiting two slow-step kinetics. The reaction temperature maydepend upon the type of polymerase which is employed. Temperaturesbetween 15° C. and 90° C., between 20° C. and 50° C., between 20° C. and40° C., or between 20° C. and 30° C. can be used.

In some cases, additives can be added to the reaction mixture that willchange the kinetics of the polymerase reaction in a manner that can leadto two slow-step kinetics. In some cases, the additives can interactwith the active site of the enzyme, acting for example as competitiveinhibitors. In some cases, additives can interact with portions of theenzyme away from the active site in a manner that will change thekinetics of the reaction so as to produce a reaction exhibiting two slowsteps. Additives that can influence the kinetics include, for example,competitive, but otherwise unreactive substrates or inhibitors inanalytical reactions to modulate the rate of reaction as described incopending U.S. Utility patent application Ser. No. 12/370,472 the fulldisclosures of which is incorporated herein by reference in its entiretyfor all purposes.

One aspect of the invention is the use of a kinetic isotope effect, suchas the addition of deuterium to the system in order to control thekinetics of the polymerase reaction in single-molecule sequencing. Insome cases, the isotope, such as deuterium can be added to influence therate of one or more step in the polymerase reaction for improvingsingle-molecule sequencing. In some cases, the deuterium can be used toslow one or more steps in the polymerase reaction due to the deuteriumisotope effect. By altering the kinetics of steps of the polymerasereaction, in some instances, two slow-step kinetics, as describedherein, can be achieved. As described in the examples below, in somecases, the addition of deuterium can be used to increase the mean pulsewidth in a single-molecule sequencing system.

Additives that can be used to control the kinetics of the polymerasereaction include the addition of organic solvents. The solvent additivesare generally water soluble organic solvents. The solvents need not besoluble at all concentrations, but are generally soluble at the amountsused to control the kinetics of the polymerase reaction. While not beingbound by theory, it is believed that the solvents can influence thethree dimensional conformation of the polymerase enzyme which can affectthe rates of the various steps in the polymerase reaction. For example,the solvents can provide affect steps involving conformational changessuch as the isomerization steps shown in FIG. 32. Added solvents canalso affect, and in some cases slow, the translocation step. The slowingof the translocation step can increase interpulse distances, and can beused in conjunction with slowing the nucleotide binding step, forexample, to obtain two slow steps in the steps in which the nucleotideis not associated with the enzyme, for instance resulting in two slowsteps in the dark phase of a polymerase reaction. In some cases, thesolvent additives can increase the interpulse distance withoutsubstantially affecting the pulse widths in single-molecule sequencing.In some cases, the solvents act by influencing hydrogen bondinginteractions. In some case, the addition of solvent can be used tochange the rate of one or more steps in the polymerase reaction. Forexample, the solvent may slow one or more steps in the polymerasereaction. By influencing the rates of various steps of thepolymerization, the solvent additives can be used, in some cases, toobtain two slow-step kinetics. The addition of organic solvents can beused, for example to increase the mean time between pulses (interpulsedistance).

The water miscible organic solvents that can be used to control therates of one or more steps of the polymerase reaction in single-moleculesequencing include alcohols, amines, amides, nitriles, sulfoxides,ethers, and esters and small molecules having more than one of thesefunctional groups. Exemplary solvents include alcohols such as methanol,ethanol, propanol, isopropanol, glycerol, and small alcohols. Thealcohols can have one, two, three, or more alcohol groups. Exemplarysolvents include small molecule ethers such as tetrahydrofuran (THF),and dioxane. In some embodiments the solvent is dimethylacetamide (DMA).In some embodiments the solvent is dimethylsulfoxide (DMSO). In someembodiments, the solvent is dimethylformamide (DMF). In some embodimentsthe solvent is acetonitrile.

The water miscible organic solvent can be present in any amountsufficient to control the kinetics of the polymerase reaction. Thesolvents are generally added in an amount less than 40% of the solventweight by weight or volume by volume. In some embodiments the solventsare added between about 0.1% and 30%, between about 1% and about 20%,between about 2% and about 15%, and between about 5% and 12%. Theeffective amount for controlling the kinetics can be determined by themethods described herein and those known in the art.

A suitable additive for obtaining a two slow-step system is the aminoacid, cysteine, having the chemical formula HO2CCH(NH2)CH2SH. Cysteinecan be added to the reaction mixture as a salt, for example, as thehydrochloride salt. Generally, the naturally occurring L-cysteine (Cys)is used. Other additives with chemical structures related to cysteinecan also be used. For example, homocysteine or any other suitablenatural or artificial amino acid having an S atom, and in particular, athiol group. We have found that the addition of cysteine can lead to anincrease in both overall yield and in accuracy of single moleculesequencing. While not being bound by theory, Cys, because of its thiolside chain and AA polar moiety may have beneficial effects on bothpolymerase and nucleotides during sequencing. An increase in the pulsewidth with the addition of Cys has also been observed. The effect couldbe different from or cumulative to that of dithiothreitol (DTT), whichcan also be added to the sequencing reaction, owing to only a single —SHfunctionality in Cys and, therefore, larger tendency to participate inintermolecular interactions. In addition, Cys may influence the analogbinding to polymerase via linking the two with hydrogen and S—S bonds.Cysteine can be added at any level suitable for improving the propertiesof the enzymatic reaction. For example, cysteine can be added at amountsgreater than about 0.1 mM, greater than about 0.5 mM, greater than about1 mM, greater than about 5 mM, greater than about 10 mM. In some cases,the cysteine can be added in amounts less than about 200 mM, less thanabout 100 mM, less than about 50 mM, less than about 20 mM, or less thanabout 10 mM. In some cases, the cysteine is present in amounts betweenabout 1 mM and about 100 mM, between about 5 mM and about 50 mM, orbetween about 10 mM and about 30 mm.

Additives such as dithiothreitol (DTT), can also be present in thereaction. In some cases, such additives, which are often used inenzymatic systems, do not directly lead to two slow-step systems, butare useful for the functioning of the enzyme during, for example,nucleic acid synthesis.

One aspect of controlling the polymerase reaction conditions relates tothe selection of the type, level, and relative amounts of cofactors. Forexample, during the course of the polymerase reaction, divalent metalco-factors, such as magnesium or manganese, will interact with theenzyme-substrate complex, playing a structural role in the definition ofthe active site. For a discussion of metal co-factor interaction inpolymerase reactions, see, e.g., Arndt, et al., Biochemistry (2001)40:5368-5375.

For example, and without being bound to any particular theory ofoperation, it is understood that metal cofactor binding in and aroundthe active site serves to stabilize binding of incoming nucleotides andis required for subsequent catalysis, e.g., as shown in steps 106 and108. Other metal cofactor binding sites in polymerases, e.g., in theexonuclease domains, are understood to contribute to differentfunctionality of the overall proteins, such as exonuclease activity.

In the context of the present invention, however, it has been discoveredthat modulation, and particularly competitive modulation of divalentmetal cofactors to the synthesis reaction can provide substantialbenefits in terms of reaction kinetics without a consequent increase innegative reaction events.

In the synthesis reaction, certain divalent or trivalent metalcofactors, such as magnesium and manganese are known to interact withthe polymerase to modulate the progress of the reaction (See, e.g., U.S.Pat. No. 5,409,811). Other divalent metal ions, such as Ca2+, have beenshown to interact with the polymerase, such as phi29 derivedpolymerases, to negative effect, e.g., to halt polymerization. As willbe appreciated, depending upon the nature of the polymerizationreaction, environmental conditions, the polymerase used, the nucleotidesemployed, etc., different metal co-factors will have widely varyingcatalytic effects upon the polymerization reaction. In the context ofthe present invention, different metal co-factors will be referred toherein based upon their relative catalytic impact on the polymerizationreaction, as compared to a different metal included under the samereaction conditions. For purposes of discussion, a first metal co-factorthat interacts with the polymerase complex to support the polymerizationreaction to a higher level than a second metal co-factor under the sameconditions is termed a “catalytic metal ion” or “catalytic metal”. Inpreferred aspects, such catalytic metals support the continued,iterative or processive polymerization of nucleic acids under theparticular polymerase reaction conditions, e.g., through the addition onmultiple bases, while in some cases, a given type of metal cofactor mayonly support addition of a single base. Such metals may be sufficientlycatalytic, depending upon the specific application.

The rate of nucleic acid translocation through nanopores under anapplied voltage can also be controlled by the binding of proteins, smallmolecules, and/or the hybridization of complimentary strands (see, e.g.,FIG. 11). The nanopore (202) physically occludes the passage of thenucleic acid strand with the bound enzyme, small molecule, orcomplementary strand (200, 204). The kinetics of nucleic acidtranslocation can be controlled by the concentration of 200 (cis side)and 204 (trans side). For example, the binding element could be: E. coliSSB, T4 gene32, Tth SSB, Taq SSB, T7 gene 2.5, or any other of the broadclass of single-stranded DNA binding proteins, which are known to beinvolved in almost every aspect of DNA metabolism. Additionally usefulare the recombinational enzymes like recA or the eukaryotic proteinsRad51 and Dmc1 because their binding properties can be modulated by theaddition of ATP, ADP, and nonhydrolyzable ATP analogs (see, e.g.,Structure and Mechanism of Escherichia coli RecA ATPase, Charles E.Bell, Molecular Microbiology, Volume 58, Issue 2, Pages 358-366).

In certain embodiments, polymerases are used to modulate the passage ofa nucleic acid strand through a nanopore. For example, it has beendemonstrated that the passage of DNA through a nanopore structure can becontrolled by the binding of Klenow fragment DNA polymerase in thepresence of varying concentrations of cognate nucleotide (SpecificNucleotide Binding and Rebinding to Individual DNA Polymerase ComplexesCaptured on a Nanopore; Nicholas Hurt, Hongyun Wang, Mark Akeson andKate R. Lieberman; J. Am. Chem. Soc., 2009, 131 (10), pp 3772-3778).Binding events can be individual and stochastic or cooperative (e.g.gene32 polymerization on single-stranded DNA) For example, see: On thethermodynamics and kinetics of the cooperative binding of bacteriophageT4-coded gene 32 (helix destabilizing) protein to nucleic acid lattices.S C Kowalczykowski, N Lonberg, J W Newport, L S Paul, and P H vonHippel; Biophys J. 1980 October; 32(1): 403-418.). In general,conditions that favor binding to the nucleic acid strand will slowtranslocation of the nucleic acid strand to the other side, andconditions that are less favorable to binding will permit relativelyfaster translocation. These factors can be modulated advantageously topromote efficient sequence detection, e.g., by allowing the reaction toproceed at a rate that provides for a desirable balance between accuracyand throughput.

One aspect of the invention is the use of processive DNA-binding enzymeto enzymatically regulate the rate of ssDNA tranlocation through thenanopore. For example, λ-exonuclease processively degrades one strand ofa dsDNA template in the 5′-3′ direction. The single-stranded part wouldsnake through the nanopore, and the excised dNMPs would diffuse away(because the ssDNA would leave no room for them to pass through thenanopore). The rate of ssDNA translocation through the nanopore wouldnow be limited by the rate of λ-exonuclease activity, which could bemodulated by Mg concentration, buffer conditions, and potentialmutagenesis of the enzyme. λ-exonuclease is described in Science. 2003Sep. 26: 301(5641):1914-8.

In some cases we can use a DNA-binding enzyme to act as a plug to thenanopore and regulate ssDNA translocation rate non-enzymatically, Forexample, Exonuclease I degrades ssDNA. However, one could use anenzymatically inactive Exonuclease I (or e.g. leave Mg out of thesolution buffer) that still binds tightly to ssDNA. Again, the unboundssDNA would snake through the nanopore, whereas the exonuclease bound tothe ssDNA would act as a plug and prevent translocation. Applying astrong enough potential can rip the ssDNA from the tightly boundexonuclease, advancing the ssDNA through the nanopore. By applying shortpulses of large potential (translocation step) separated by periods oflower potential (allows rebinding of exonuclease), then one can pull thessDNA through the nanopore in steps, for example one base at a time. Therate and duty cycle of the pulses could be altered to optimize thetranslocation rate and measurement duration.

For this embodiment, DNA binding proteins other than an exonuclease canbe used. For example, a DNA polymerase locked in the closed state (e.g.by having calcium but no magnesium in the solution) may be used. In thiscase, the dsDNA primer can get peeled off one base at a time as the highpotential pulse pulls the ssDNA through the pore.

Alternatively, a histone can be used. 146 base pairs at a time of dsDNAgenerally wrap around a histone complex like a spool. As above, thehistone would act as a stop to the nanopore. High potential pulses wouldunravel the spool one base at a time. As with the polymerase, one of thetwo strands in the dsDNA would still have to be peeled off by thenanopore, which only allows ssDNA to pass through.

Once aspect of the present invention is to use a processive polymerasesuch as Phi29 with a nanopore. The polymerase is applied on the upstreamside of the nanopore, as well as the DNA template to be sequenced andprimer, if any. dNTPS are added to the solution at a concentration thatallows a sufficiently long time between base incorporation events tofacilitate accurate readout from the nanopore for each base position.The use of a processive enzyme allows the baseline nanopore signal to befree of disturbance caused by the binding and unbinding of polymerase.Another aspect of the present invention is to use a strand displacingenzyme and to thread the displaced product rather than the templatethrough the nanopore. In this way, the direction of DNA motion is in thesame direction as the applied electric force. This allows increasedreadlength, reduction in buildup of extraneous DNA at the upstream sideof the pore as well as other problems. Another aspect of the inventionis to use an enzyme with two or more slow steps in the translocationstep. This would allow for decreased incidence of events that are tooshort to be reliably detected. An additional advantage of using thedisplaced product rather than the template, is that the template can bemaintained in a double-stranded state, thus increasing the stability ofthe template, and allowing for longer readlength.

One embodiment for controlling translocation during sequencing isillustrated in FIG. 12. A DNA polymerase enzyme with strand displacementis used to create a single strand of DNA which is then translocatedthrough the nanopore. The circular template will result in a replicationof the same sequence multiple times (rolling circle amplification),allowing for higher accuracy. The reagents necessary for performing DNAsynthesis, including nucleotides and cofactors are provided on the cisside of the nanopore in order to support synthesis.

Another embodiment for determining sequence information about a templatepolymer by controlling translocation is shown in FIG. 13. A DNAdependent RNA polymerase is used to produce an RNA transcript, which istranslocated through the channel and sequenced.

Electronic Control of Translocation Rate—Molecular Braking

One aspect of the invention is the control of translocation byelectrical processes. In other embodiments, translocation of a molecule(e.g., a polynucleotide) through a nanopore can be controlledelectrically. For example, and with reference to FIG. 14, one skilled inthe art will realize that electric fields within the supporting membrane(100) and transverse to the nanopore (101) can be used to manipulate asingle-stranded DNA molecule (102) because the DNA backbone phosphatesgenerally carry a net negative charge. In essence, the field attractsDNA toward the positive terminal and pulls the DNA against any physicalbarrier. Steric interactions (i.e., microscopic friction) with thebarrier reduce the kinetic energy of the translocating DNA, initiallyinduced by an additional bulk solution field (103), through conversionto heat. This effect is termed “Molecular Braking,” and thenanostructure that is the “Molecular Brake” includes, but is not limitedto, the supporting membrane (100), transverse electrodes (which may ormay not be the supporting membrane; fabrication discussed below), andthe nanopore (101).

Optionally, the transverse electric field can be either AC or DC.Optionally, the Molecular Brake can be applied when the functionalcurrent readout of the DNA translocation is either through additionalbulk solution electrodes (104) or through nanogap detection, i.e.,through a tunneling current between electrodes embossed in thesupporting membrane (105), as shown in FIGS. 15A and 15B.

Several means of fabricating such a Molecular Brake are available to oneskilled in the art, e.g., on an insulating substrate (106), growing athin metal film and dividing into two pads separated by a very thin gap(107; similar to Liang and Chou, Nano Letters, 8:5 1472 (2008)),evaporating on an insulating “cover” (108), and fabricating a nanoporethrough the channel (109) by, e.g., SEM drilling or transverse electronbeam ablation lithography, examples of each of which are shown in FIG.16.

Optionally, and with reference to FIG. 17, the nanopore can have acylindrical profile (110), hourglass profile (111), conical profile(112) or an elliptical cylindrical profile (113), and in preferredoperation would have a minimal transverse diameter of less than 3 nm andlength of less than 500 nm. Although shown as having straight walls, thewalls may also be tapered or otherwise shaped while retaining theoverall cylindrical, conical, hourglass, or elliptical cylindricalprofile. Further, in certain preferred embodiments the hourglass profilewould be used as this profile reduces the steepness of the entropicbarrier as DNA enters the pore, and the bulk solution voltage drop fromcis to trans occurs over just a few nanometers at the tightestconstriction of this pore (see, e.g., corner et al., Biophys. J.96:593-608, (2009)). Further, the location within the nanopore at whichdetection occurs may be positioned at the center of the nanopore, or maybe nearer to either the cis or trans end of the nanopore, and isoptionally located at a point in the nanopore that is constrictedrelative to other positions within the nanopore.

Beyond Molecular Brakes, it is possible to use a stack of conductingpads that are electrically addressable to convey the DNA in lock stepthrough the nanopore. Local inhomogeneities in the DNA chargedistribution enable this such that even if the conducting layers arethicker than the phosphate backbone spacing, active transport may stillbe possible, termed the “Molecular Sidewalk.” When charge variation isnaturally present along the DNA target template, and this is constrainedlaterally in an alternating potential, e.g., by a nanopore through astack of differentially-charged plates, then DNA regions willpreferentially localize within that potential and may be held againstthermal energy. If that periodic potential is translocated from cis totrans, then the DNA that is caught within that potential will betransported in lock-step. Further, should the DNA encounter symmetricenergy barriers for moving cis versus trans as the Molecular Sidewalkpotential sweeps to trans, the bulk solution voltage will break thatsymmetry and may induce motion to trans. Shown in FIG. 18 is a DNAmolecule (one position marked with an “x” for clarity) being transporteddown through the pore with alternating fields.

The function of the Molecular Sidewalk can occur by eitheraforementioned detection modes. The fabrication architecture ofMolecular Brakes can be extended to multiple layers of conducting padsthat are electrically isolated and individually addressable. (See, e.g.,FIG. 19.)

Optionally, the Molecular Sidewalk may also be combined with brakingmethods including but not limited to Molecular Braking. In oneimplementation, a cis-side Molecular Brake is combined with a trans-sideMolecular Sidewalk. One skilled in the art realizes that DNA bunchingmay occur for the Molecular Sidewalk if not carefully implemented, dueto, e.g., sequence context variation that causes a given region of thestrand to localize to a local potential minimum. This combination mayyield entropic and enthalpic stretching of the DNA as the MolecularSidewalk pulls the DNA through the pore, with the Molecular Breakretarding that motion. Optionally, a nanogap detector could be locatedbetween the Molecular Brake and Molecular Sidewalk in the supportingmembrane, where the DNA may be optimally positioned for detection.Optionally, braking may be achieved with DNA binding moieties includingbut not limited to proteinaceous compounds (e.g., RecA or Gene 32) orshort nucleic acid polymers (i.e., random or nonrandom sequences ofvarious lengths that anneal to the target template and must bedissociated from said template by force as translocation occurs), asdescribed above.

In certain preferred embodiments, the per base translocation ratethrough all devices or combinations of devices would be between 100 Hzand 100 MHz.

Electronic Control of Translocation Rate—Molecular Iris

Even with the ability to differentiate the distinct current-basedsignals (“signatures”) produced by passage of the four different basesthrough the nanopore, single-molecule sequencing with nanopores isfundamentally challenged by the ability to detect and characterizehomopolymer regions of a target template. The primary reason for this isdue to the identical signals produced for subsequent positions of thesame base, and difficulties quantifying how many of the same signals arebeing detected. In certain embodiments of the instant invention, anapproach, termed the “Molecular Iris,” is used to increase systemresolution by making base-wise translocation through the nanopore moreclock-like, thereby promoting individually detectable current signaturesfor every base translocation through the nanopore.

This approach is analogous to a molecular-scale ratchet and pawl systemwhere the pawl tension is very stiff relative the energy that is movingthe ratchet (e.g., high energetic barrier to move forward; much, muchhigher barrier to move backward). Without being bound by any particulartheory of operation, the general implication is that a given position ofthe ratchet will be sampled on a longer time scale than the overalltimescale associated with translocation. For the nanopore system, apolynucleotide passes through the nanopore and represents the ratchetwith the bases as teeth. The pawl in this system is an element on thepore wall that interacts with the bases, e.g., intercalates between thebases. Interaction of the pawl with a given base causes translocation toeffectively pause at that base, allowing the current signature of thebase to be accurately and individually detected. As such, each baseposition can be sampled for a higher duty cycle relative overallbase-to-base translocation due to the presence of the pawl.

An embodiment of this aspect of invention is shown in FIG. 20. A keyfeature of the membrane (100) supported nanopore (101) system is thepawl (102), or set of pawls (103), that are inside the nanopore barreland interact with single-stranded polynucleotide (e.g., DNA) (104).Because these device elements restrict motion through the barrel bypartially closing it off, we term this system the “Molecular Iris.”

The multi-pawl case is illustrated in FIG. 21. For the multi-pawl case,the closed (104) state is generally the state at which the nanoporebarrel is most restricted and the open (105) state is generally thestate at which the nanopore barrel is least restricted. In certainembodiments, the closed configuration has all pawls directed toward themolecule passing through the nanopore (e.g., pointed inward), and theopen configuration has all pawls directed away from the molecule (e.g.,pointed upward or downward, or otherwise retracted away from themolecule.) Optionally, the pawls may move in concert or independently.Various other embodiments of open and closed configurations will beclear to those of ordinary skill in the art.

Pawls may include but are not limited to nucleic acids or amino acids,either in side chain or polymer forms, small-molecules such as ethyleneglycol or solid state materials with modulated physical properties(e.g., piezoelectric material that expands/contracts in an externalfield). Pawls may be embedded in either a synthetic nanopore, biologicalnanopore, or a chimera of both.

One skilled in the art will recognize that biological nanopores (e.g.,multi-subunit nanopores, including but not limited to naturallyoccurring alpha hemolysin and MspA) or subunit concatemers (in which theDNA monomer code is copied and concatenated, resulting in a singlepolypeptide for the entire protein) can be mutated for attachment ofpawls. Such methods include mutagenesis to add or substitute extraresidues that would interact with the DNA (including but not limited topolar residue phenylalanine, tryptophan and histine, or charged residuesaspartate, glutamate, lysine, arginine, or histidine), residue mutationto cysteine for disulfide linking chemistry to proteinaceous or solidstate pawls, or other methods. One particularly useful approach is toincorporate unnatural amino acids into the protein nanopore order toproduce the molecular iris. In this way, the desired chemical propertiescan be engineered into the protein, e.g. in a repeated subunit, withouthaving to perform reactions on the protein after it is formed. Methodsof incorporating non-natural amino acids are well known in the art.Fusion proteins can also be used to produce such structures.

There are several advantages of including a pawl or pawl complex in ananopore over standard nanopore sequencing. (1) A pawl that interactsstrongly with each base may confer extra sensitivity and specificity tothe current flowing around that base, including but not limited tohydrophobic ring stacking (e.g., between the base and a tryptophan pawl)or steric effects (e.g., between the base and a proline). (2) Amulti-pawl complex means that several elements must move to allow DNAtranslocation, which is likely to render transport more uniform in speed(i.e., more clock-like), though one skilled in the art will realize thatoverall speed can additionally or alternatively be controlled by poresize and driving voltage. (3) Because the pawl must move to step fromone base to the next (i.e., the Molecular Iris goes from a closed stateto open and back to closed for a single translocation), a significantcurrent may be discharged even during homopolymer sequencing, which canbe keyed upon for base calling of sequential nucleotides having the samebase composition.

Multiple Stage Nanopore Sequencing

One aspect of the invention involves using multi-staged nanopores forobtaining polymer sequencing information. In nanopore DNA sequencing,base calling is performed by detecting the current blocking events asssDNA or single dNMPs translocate through the pore (often either amodified alpha-hemolysin protein pore or a solid-state pore). See e.g.Nature Biotechnology, 26 (10):1146-1153, (2008). A combination of theamplitude and the duration of the current block is used to distinguishthe four nucleotides from one another. However, the amplitude of currentblockage for each nucleotide has a Gaussian distribution, and thedistributions from each of the four nucleotides can overlapsignificantly (more or less so depending on the solution conditions),increasing the likelihood of miscall errors. A means of performingconsensus calling in order to reduce this error source is describedbelow.

If one nanopore embedded in a membrane that separates two compartmentsis considered one stage, then by having more than one membrane, we canconcatenate multiple stages. For example, once the analyte (e.g. ssDNAor dNMP) has passed through the first stage nanopore, it could then passdirectly through a second nanopore, or a second stage of measurement. Ifthe current blockage through each stage is statistically independent(e.g. noise is dominated by random diffusion and the channels arenarrow), then one can compare the two reads and perform consensus basecalling based on the two measurements. The multistage nanopore devicesof the invention can have 2, 3, 4, 5 or more stages. The number ofstages can be generalized to N stages (N independent sets of nanopores)to further improve base calling accuracy to the required level.

In one embodiment, each stage's electrodes are not shared. Thus, for Nstages, there would be a total of 2N electrodes (one above and anotherbelow each stage).

In another embodiment, adjacent stages share an electrode (e.g. Stage 1has an electrode on top, and then its bottom electrode serves as the topelectrode for Stage 2, which would also have its own bottom electrode).Thus, for N stages there would be a total of N+1 electrodes. An examplethree-stage system is shown in FIG. 22 (electrodes are not shown).

In one embodiment, the sequencing strategy involves attaching anexonuclease to the nanopore, cleaving dNMPs from dsDNA, and detectingthe passage of these dNMPs through the nanopore, then for the multistagenanopore device described herein would only have an exonuclease attachedto the first stage's nanopores, but would obtain multiple opportunitiesto measure the monomers.

Another advantage of this technique is that it can reduce the number ofmissed pulses, since each nucleotide could be directed to pass through apore several times and thus have several opportunities to be measured.

This multi stage devices and methods of the invention could be used withsolid-state nanopores, protein nanopores, and hybrid protein/solid-statenanopores. Furthermore, a similar technique could be used with atunneling current measurement scheme.

Each stage can comprise multiple nanopores, e.g. each state can be alayer of nanopores, each with 2-10-100, 1000 or more nanopores. Thenumber of pores in the various layers can be coupled such that flowcontinues through only one set of pores. In other cases, the pores canbe decoupled. In some cases, current measurement made at each stage, inother cases, measurements made only after multiple stages.

One embodiment comprises a linked complex of two or more nanopores inseries—and one electrical measurement system. Distribution of currentblockage duration will be the convolution of the exponentialdistributions of those for each individual nanopore. In someembodiments, each of the N nanopores could be different—e.g. moreeffective at distinguishing particular bases. These structures can becreated, for example, by genetically engineering the multiple nanoporesas fusion proteins. Alternatively, the individual nanopores can belinked, e.g. hydrophobically. In some cases “terminating” nanopores canbe added to control nanopore concatenation. In some cases, specific topand bottom terminating nanopores can be used to control nanoporeconcatenation.

Use of Tunneling Current and Multiple Stages

One aspect of the invention is the use of tunneling current andmulti-staged nanopores. It has been suggested that the ability todiscriminate between bases can be enhanced by using a tunneling currenttechnique and by forming base-specific hydrogen bonds between thenucleotide being detecting and a chemically modified pore or tunnelingcurrent probe. This has been described for use in conjunction with atransverse tunneling current measurement. For example, the probe couldbe functionalized with one of four nucleotides (e.g. cytosine), and thenthe tunneling current would be greatly enhanced when the complementarynucleotide (e.g. guanine) passes through the pore. See references Proc.Natl. Acad. Sci. USA 103, 10-14 (2006); Nano Lett. 7, 3854-3858 (2007).

A potential disadvantage of this technique, however, is that it wouldrequire four readers (each functionalized with a distinct nucleotide)sequencing duplicate strands in synchrony, a difficult task to achieveNature Biotechnology, 26 (10):1146-1153, (2008). We have discovered thata multistage nanopore system of the current invention can address thisissue. Instead of four readers sequence four duplicate strands, thedevice of the current invention would have multiple stages of readers,for example, four stages of readers wherein each is functionalized witha distinct nucleotide for sequencing the same strand. FIG. 23 (a) showsa schematic drawing of a multi-staged tunneling current measurementsystem. In this case, the multi-staged tunneling current nanopore systemconsists of all solid-state nanopores or of hybrid protein/solid statenanopores.

FIG. 23(b) shows an alternative multi-stage tunneling embodiment havingone channel with several transverse tunneling measurement stages. Forexample, the device can comprise, one long solid-state nanopore thatcontains 4 tunneling current probes along its length, eachfunctionalized with a different nucleotide.

Use of Tunneling Current

One aspect of the invention involves the measurement of tunnelingcurrent to determine sequence information using a multiplex solid statearray of nanopores. Given typical drive voltages of a few hundred mV,typical ionic currents flowing through a <3 nm diameter nanopore are inthe picoamp or tens of picoamp range. Using state-of-the-art detectors,the detection of such small currents can generally be accomplished with˜kHz bandwidths. For example, events (e.g. nucleotides traversing thenanopore for sequencing applications) can be detected faithfully wheretheir duration is on the order of milliseconds.

Since nucleotides under a 120 mV potential can traverse analpha-hemolysin nanopore in microseconds, Nature Biotechnology, 26(10):1146-1153, (2008), one solution has been to insert an adaptormolecule into the alpha-hemolysin nanopore in order to slow down thenucleotide traversal, JACS, 128:1705-1710 (2006). Another solutionsuggested in the literature has been to instead measure the transversetunneling current between 1 nm diameter probes situation across ananopore Nano Lett. 5, 421-424 (2005); Phys. Rev. E 74, 011919 (2006);J. Chem. Phys. 128, 041103 (2008); Nano Lett. 6, 779-782 (2006);Biophys. J. 91, L04-L06 (2006). The advantage of this technique is thattunneling currents can be in the nanoamp range Nano Lett. 7, 3854-3858(2007), which would enable state-of-the art detectors to measure themicrosecond timescale events, such as the translocation of nucleotidesthrough unmodified pores.

Descriptions of tunneling current nanopore systems in the literaturegenerally describe solid-state nanopores, since these can be fabricatedalong with the nano-electronic components required for tunneling currentmeasurements. Fabricated nanopores, however, can also have a largevariation in size, shape, orientation, surface chemistry, etc. betweenindividual nanopores. This has been noted in a review article as achallenge for tunneling current nanopore sequencing, since the tunnelingcurrent is very sensitive to orientations of and distances between theelectrodes and the nucleotides to be detected Nature Biotechnology, 26(10):1146-1153, (2008). One literature proposal is to use carbonnanotubes as a nanopore, as carbon nanotubes have a reproduciblesize/shape and bind nucleotides in a specific manner Nano Lett. 7,1191-1194 (2007).

One aspect of the invention comprises creating a hybridprotein/solid-state nanopore for tunneling current nanopore sequencing.The use of protein nanopores, such as alpha-hemolysin, for DNAsequencing has been well documented in the literature JACS,128:1705-1710 (2006). A great advantage of protein nanopores is thateach nanopore is very similar to every other nanopore, yielding anhomogeneity in nucleotide orientation/position between each event ineach different nanopore. Furthermore, protein nanopores can readily bemutated or hybridized with a linker molecule in order to enhance manyproperties of the nanopore sequencing system (e.g. increase thenucleotide residence time within the pore, or enhance discriminationbetween nucleotides). Tunneling current measurements with standardprotein nanopore sequencing systems are impossible, though, becauseprotein nanopore are generally embedded in a lipid bilayer JACS,128:1705-1710 (2006). In the current invention, the surfacefunctionalized solid-state scaffolding in which the protein nanopore isembedded enables integration with tunneling current electronics. The useof tunneling current can be particularly useful when combined with themultistage nanopore designs described above.

Sequencing Using Combined Polymerase/Exonuclease Activity

One aspect the invention utilizes a polymerase/exonuclease pair to pushthen pull back a DNA strand in the nanopore. In some cases, two separateenzymes can be used, in other cases, the enzyme activities can be in asingle enzyme. For instance, in the same enzyme such as Phi29 DNApolymerase. One method for carrying out the invention comprises: 1)adding nucleotides and making use of the polymerization process topush/pull the dna through the nanopore for detection, 2) Removingnucleotides through a wash step, allowing exonuclease activity to kickin and push/pull the dna in the opposite direction of the polymeraseactivity, 3) Repeating step 1 and cycling. Adjusting the relative ratesrate of exonuclease or polymerase speed can be achieved throughmutations such as those described herein for polymerases. The relativerates can also be controlled by reaction conditions, such as bycontrolling the concentration of the nucleotides in solution availablefor the polymerase. At high nucleotide concentrations, the polymerasewill proceed relatively rapidly, and at low nucleotide concentrationsthe polymerase will proceed more slowly. In addition, if the desire isto read a cleaved moiety, it has been suggested to use an exonuclease tocleave off a base, which then passes through the pore and detected. Theinvention disclosed here uses a polymerase/exonuclease pair to firstpolymerize, and use a modified cleaved phosphate group as the detectionmoiety. Then, after one or more bases, activate exonuclease activity anddetect the cleaved base. This allows not only the ability to performmultiple reads on the same strand of DNA, but allows different detectionmoieties. This method of incorporating both polymerase and exonucleaseactivity can improve overall sequencing accuracy.

Nanopore-in-Well

One aspect of the invention comprises placing the nanopore within a wellstructure. Single-molecule nanopore DNA sequencing schemes have beendescribed in which a nanopore is embedded in a flat or nearly flatmembrane. An exonuclease is fixed adjacent to the nanopore. As theexonuclease chews up double-stranded DNA, dNMPs are released. A voltageapplied across the membrane pulls the released dNMPs through thenanopore, where they are detected and differentiated from one anotherusing current blockage amplitudes, nanopore residence times, or othermetrics. See Clark et al., Nature Nanotechnology 4(4), 265-270 (2009).

A problem with this approach is that there is a probability that thedNMP will diffuse away into the bulk solution before the applied voltagecan pull it through the nanopore. This situation would lead to a missedbase call if the next dNMP to be released by the exonuclease is pulledthrough the nanopore before the diffusing dNMP makes its way back to thenanopore opening. Furthermore, this dNMP might later diffuse back to thenanopore opening or into a different nanopore's opening (in the case ofparallel nanopore sequencing), leading to a false-positive base call.

One aspect of the invention is a structure in which the nanopore is heldin a well structure rather than on a relatively flat plane in order toreduce the likelihood that, upon release by an exonuclease, a dNMP willdiffuse into the bulk solution. Thus, this aspect of the invention canincrease the fidelity with which a dNMP is pulled through the nanoporeimmediately upon release by the exonuclease. In this invention, thenanopore is depressed within a well (see FIG. 24(b)). The well decreasesthe probability of the dNMP diffusing into bulk solution in two ways.

While not to be bound by theory, we believe that using the wellstructures of the invention improve accuracy both by entropy and byenthalpy. Through entropy: on the flat membrane, if the dNMP diffusesfirst in the z-direction then it will go directly into the nanopore. Itwill stay in the nanopore despite a subsequent diffusion of e.g. 100units in the x- or y-direction. However, if it first diffuses in the x-or y-direction by e.g. 100 units, then it has already diffused away fromthe nanopore opening, and it will not enter the nanopore upon asubsequent z-direction diffusion event. This asymmetry may delay thedNMP from passing through the nanopore before the next dNMP does so.However, if the nanopore is depressed in a well, the asymmetry is not assevere. If the dNMP first diffuses in the x- or y-direction by e.g. 100units, it may bounce of the wall of the well and end up positioned overthe nanopore opening. A subsequent diffusion event in the z-directionwould result in the dNMP passing through the nanopore and beingdetected.

Through enthalpy: in the case of the flat membrane, the current density“field” lines fan out in a roughly spherical shape, and the densitydecreases rapidly as the radial distance from the nanopore centerincreases. Thus, if the dNMP does diffuse away from the nanopore centerand against the energy barrier (through thermal fluctuations dependingon the thermal Boltzmann factor kBT) by e.g. 100 units, the energybarrier for the particle to move e.g. another 100 units away from thenanopore is lower, and thus it is even easier for it to diffuse evenfurther away. On the other hand, the current density “field” lineswithin the well are parallel and maintain the same density until theopening of the well is reached, upon which the lines fan out as before.Within the well, the energy barrier for the particle diffusing e.g. 100units away from the nanopore is not decreasing as the dNMP gets furtherand further away (but remains inside the well). Thus, within the well,the particle is less likely to diffuse against the energy barrier due tothe applied voltage. FIG. 24(b) illustrates a nanopore in a wellstructure of the invention. In some embodiments, the height to width ofthe well is about 1 to 1, about 2 to 1, about 3 to 1, about 5 to 1,about 10 to 1, or more than 10 to 1. In some cases the average heightand average width is used. The shape of the well structure can be anysuitable shape.

One aspect of the invention comprises the use of a magnetic orparamagnetic label onto the polymer to be sequenced, and using amagnetic field to control the translocation of the polymer through thenanopore. In some cases, the magnetic field will be used in conjunctionwith drive electrodes. In some cases the magnetic field alone can beused to translocate the polymer. Where only the magnetic field is usedto translocate, the system can be simplified because no driveelectronics are needed, and the currents required for electronicallydriving the molecules through the pore are not required.

AC Field Dielectrophoresis

One aspect of the invention is the incorporation of AC dielectrophoresisto assist in transporting the molecules of interest through a nanopore.In some sequencing methods, e.g. utilizing exonucleases as describedabove, there is a probability that a molecule of interest, such as adNMP will diffuse away into the bulk solution before the applied voltagecan pull it through the nanopore. This situation would lead to a missedbase call if the next dNMP to be released by the exonuclease is pulledthrough the nanopore before the diffusing dNMP makes its way back to thenanopore opening. Furthermore, this dNMP might later diffuse back to thenanopore opening or into a different nanopore's opening (in the case ofparallel nanopore sequencing), leading to a false-positive base call.

It is known that DNA can be moved or sorted by dielectrophoresis (thegradient of an electric field, such as that through a nanopore under anapplied potential, can apply a force to a polarizable material). SeeElectrophoresis, 23 (16): 2658-2666. Furthermore, there are peaks in thefrequency spectrum at which DNA is most highly polarizable and at whichdielectrophoresis is most effect. The same effect will likely apply toindividual dNMPs. By applying a potential with a DC offset (for theelectrophoretic component of pulling a charged particle through thenanopore) and an AC component at a peak in the dielectrophoreticfrequency spectrum of an individual nucleotide, the movement of anucleotide through the nanopore is enhanced.

This technique would reduce errors in nanopore sequencing by enhancingthe probability that a dNMP gets pulled directly through the nanoporeafter excision by the exonuclease. This technique may also be applied tothe method of nanopore sequencing in which a ssDNA is pulled through thepore, and it would enhance the probability that the ssDNA would bepulled successfully all the way through the pore.

In this embodiment of the invention, the nanopore sequencing takes placewithout any DC component of the applied electric field. This isadvantageous because DC drive can result in either electrolysis of wateror the dissolution of metal ions at the drive electrodes, both of whichstand to degrade the performance of the system unless the driveelectrodes are far from the detection center. In this embodiment, themotive force to preferentially drive the DNA in one direction isdielectrophoresis. A local zone of constricted electric fields isestablished and because of the large dipole moment of DNA over a widerange of applied frequencies, the DNA molecules feels a net forceattractive towards the high-AC-electric-field region of the fluid. Thishigh AC electric field region can be implemented either through thepresence of an electrode or through a constriction in the fluid paththat obliges the AC electric field lines to converge due to the equationof continuity. See Chou et al. Biophysical Journal, 83, 2179-2179(2002).

This zone of high AC field is positioned proximal to the detectionnanopore such that a DNA molecule traversing the nanopore is likely tohave one end fall into the potential well of the high AC field region.When this happens, there will then be a net force causing the moleculethread through the nanopore at a constant rate, Turner S W, Cabodi M,Craighead H G, Phys Rev Lett. 2002 Mar. 25; 88(12), thus allowingreadout of the DNA molecule along its length. To initially load themolecules, a DC drive force is required, however it need only be for aduration long enough to thread the molecule. For this purpose a loadingpulse is applied for a duration long enough to cause a nearby DNAmolecule to thread the nanopore, but not long enough to exhaust thenon-electrolytic (and non-dissolving) capacity of the nearby electrode.This force would bring the molecule into the capture region of thedielectricphoretic trap, at which point the AC field is applied and theDC charge displacement can be slowly reversed at a rate that does notoverwhelm the dielectrophoretic trap. In this way the net charge on theelectrode is returned to neutral without unthreading the molecule. Thesensing of the nanopore conductance is performed by measuring thecurrent voltage relationship in the AC regime.

Another aspect of the invention is methods to measure nanoporeconductance during a changing electric field environment without losingfidelity. In some operating regimes, the applied frequency must be lowcompared with the base transit time. For example at some ionicstrengths, DNA is known to have a large dipole moment at 400 Hz, whichis high enough to avoid electrolysis for many practical electrodedesigns, but is much slower than the base transit time, which means thatAC techniques for measuring the effect of one base on the conductancecannot be used. To overcome this, the measurement is performed in aquasi-DC mode in which the instantaneous field is known because of thepredictable dependence of the AC field with time.

In another embodiment the instantaneous drive voltage is measured toallow explicit comparison of the current with the instantaneous voltage.In this mode, groups of bases are read in a group and then re-read inthe opposite direction. At the points in time when the instantaneousfield is low (near the turn-around times) the system loses resolution onthe bases, potentially creating zones of confusion. Thus, one aspect ofthe invention is a selection of an amplitude an frequency that arrangeit so that the zones of confusion resulting from field reversal do notcoincide on the DNA sequence to create blackouts of information, butrather each subsequent thrust places a zone of confusion in a regionthat has been unambiguously covered by a prior thrust or will be coveredby a future thrust. It is an aspect of the invention that much of thesequence will be covered more than once, allowing for error correctionon the sequence even from a single molecule. This aspect of theinvention can be appreciated also using a combination of DC and ACfields.

Field Modulation—Noise Reduction

In one aspect of the invention, the electric field across the pore ismodulated at a specific frequency or set of frequencies, and themeasurement electronics are tuned to be sensitive to signalscorresponding to the modulation frequency or frequencies. The modulationfrequency will generally higher than the frequency at which the measuredevents are occurring. In some cases the modulation frequency is 5 times,10 times, 100 times, or 1000 times the frequency at which the monomersare being detected through the pores. In some cases, a frequencymodulation on top of the driving field e.g. at a frequency 10× orgreater than the applied field is provided. By coupling the detectionfrequency to a perturbation frequency in this manner, higher sensitivitycan be achieved by filtering out unwanted current fluctuations.

Polymerase in Microchannel

One aspect of the invention comprises measuring sequence informationabout a nucleic acid polymer by incorporating a polymerase enzyme withina channel. For the purposes of the devices and methods described above,the channel comprising the polymerase can be seen to act as a nanometerscale aperture. The requirements of the channel for this embodiment canbe different than that for other embodiments described herein. Insteadof using a very narrow and short nanopore (on the order of a few nm indiameter and length), we use a nanochannel that can be longer and can bea few nm to tens or hundreds of nanometers in diameter.

In one embodiment, a DNA polymerase-DNA template construct is placedinside the nanochannel. Nucleotides in solution are labeled on theirterminal phosphates with any type of label (a few nm to hundreds of nmin diameter) that will cause a detectable change in current flow withinthe nanochannel (e.g. metal nanoparticles, dielectric nanoparticles,highly charged nanoparticles or biomolecules, large polymers ordendrimers, etc.). A voltage is applied across the axial length of thenanochannel, and the current is measured. When the polymeraseincorporates the labeled nucleotide into the growing DNA strand, thecurrent will either increase or decrease in a detectable way for theduration of incorporation (several milliseconds—hundreds ofmilliseconds). This signal can be distinguished from diffusion oflabeled nucleotides into and out of the nanochannel because such eventswill be much shorter in duration (tens to hundreds of microseconds). Insome embodiments, after incorporation, the polymerase cleaves andreleases the label from the nucleotide with the cleavage and release ofthe phosphate.

In addition, the impact on conductivity of a transiently immobilizedlabel can be made to be different to the conductivity change broughtabout by the presence of a freely diffusing (and drifting) label. Insome embodiments of the invention, labels are chosen whose conductivity,when mobile, is matched with the conductivity of the surrounding medium,but which when immobilized can cause either an increase or decrease inthe conductivity of the channel, depending on the buffer conditions, themolecular volume, the permeability of the label molecule structure, andother parameters. In this way, the freely diffusing molecules areinvisible in the conductivity signal, because they participate inelectrical conduction to the same degree as the surrounding medium. Inother embodiments, the labels are chosen so that freely diffusing labelsinduce an increase while an immobilized label causes a decrease inconductivity. In other embodiments the free labels decrease conductivitywhile the bound labels increase it. By providing an opposite sign of theinfluence it is possible to differentiate free from bound label whilebeing able to see both. In other embodiments, the labels produce adifferent impact on conductivity before and after they have beendisconnected from their analyte molecule. In this mode it is possible tovisualize all three phases of the cycle: diffusive entry into thechannel, binding in the molecule, and then release of the label afternucleotidyl transfer. In this way, productive vs. unproductive bindingcan be distinguished. In some embodiments, the connected label isinvisible by conductivity matching, while the cleaved label is visible.In another embodiment, the free label is detectable while the cleavedlabel is invisible due to conductivity matching.

The detection of events for this aspect of the invention can beinherently different from other nanopore sequencing methods, because thedetected signal is providing information about the time in which anucleotides unit is bound within the active site of an enzyme.

The diffusive mobility of a free label can be different than that of alabel still attached to a nucleotide. Since this technique useselectrical detection, the sample rates of measurement can be tens tohundreds of kilohertz. Thus, a branching event (nucleotide istemporarily incorporated, but then dissociates without the label beingcleaved) could be distinguished from a true incorporation: a branchingevent will have the same slope (in a current vs. time graph) at thebeginning and end of a pulse, whereas a true incorporation would have asteeper slope at the pulse end, when the free label diffuses awayquickly.

Any suitable type of label (molecule, nanoparticle, quantum dot) of anyshape (sphere, ellipsoid, pyramidal, etc.) that would yield a detectablechange in the current signal could be used. Any shaped nanochannel couldbe used (conical, cylindrical, box-like, etc.). The polymerase could bein the middle of the nanochannel, at either entrance, or disposed at anysuitable place within the nanochannel. See e.g. Williams et al. U.S.Pat. No. 7,625,701B2.

Attachment of Template to the Nanochannel

One aspect of the invention comprises performing nanopore sequencing ina system in which a template polymer is attached to the nanochannel. Inone suggested method of nanopore DNA sequencing, see e.g. Clark et al.,Nature Nanotechnology 4(4), 265-270 (2009), an exonuclease is coupled toa protein nanopore (e.g. alpha-hemolysin), either as a fusion protein orthrough a linker molecule. The exonuclease degrades double-stranded orsingle-stranded DNA base by base, and then an applied voltage pulls thediffusing dNMP through the nanopore (the exonuclease should be in closeproximity to the mouth of the nanopore to decrease the likelihood thatdNMPs will diffuse away). A drop in the current through the nanopore asdNMP passes through serves to identify the dNMP. It is challenging tocreate such a complex without compromising characteristics of theexonuclease, the protein nanopore, or both. Even with such a complex,read-lengths would generally be limited by the processivity of theexonuclease because the read ends once the exonuclease lets go of thetemplate strand of DNA.

This aspect of the invention comprises a protein nanopore that has alinker molecule to attach dsDNA or ssDNA (see FIGS. 25A-D). For example,the protein nanopore can be fused to a streptavidin that will capturebiotinylated DNA. Other DNA linking techniques known in the art can beused. In the method of this invention, an exonuclease can bind to thetemplate DNA strand and begin cleaving off dNMPs, which are pulled bythe applied potential through the protein nanopore. An advantage of thistechnique is an increase read-lengths beyond the processivity of theexonuclease, because if one exonuclease falls of the DNA template, thetemplate is still bound to the same nanopore. Another exonuclease in thesolution can then rebind the DNA template and sequencing can continue.Read-lengths are thus only limited by the length of the DNA template.Furthermore, a fusion/linked complex of exonuclease/protein nanoporedoes not have to be constructed.

FIG. 25(A) shows a double stranded DNA template molecule attached to aprotein nanopore held within a membrane. Here, an alpha hemolysinprotein nanopore suspended in a lipid bilayer is used. In some cases thetemplate nucleic acid will be a single stranded nucleic acid such assingle stranded DNA. The template DNA is attached on the cis side of thenanopore with a linker, and the an exonuclease is acting on the templateDNA to excise dNMPs. The excised dNTPs are driven through the nanoporeand detected as they pass through the pore. Having the DNA template nearthe nanopore increases the likelihood that the dNMPs will be effectivelytransported through the nanopore. In FIG. 25(B), the DNA is attached tothe nanopore in two locations on the DNA strand. Here, the template is adouble-stranded DNA, and one of the strands is attached with linker toopposite sides of the nanopore by linker molecules; one linker attachedto the 5′ end and the other linker attached to the 3′ end of the DNAstrand. By attaching both ends of the template DNA, the dNMPs areexcised near the nanopore throughout the exonuclease cleavage of thestrand. Attachment at two locations on the DNA template can be usefulfor the sequencing of long DNA template molecules. FIG. 25(C) shows theattachment of the DNA template to a solid state nanopore. FIG. 25(D)shows the attachment of the DNA template to a hybrid solid state/proteinnanopore.

While in this aspect of the invention the exonuclease may not be in asclose proximity to the protein nanopore as it is were it fused or linkedto the nanopore, it will generally be close enough. Due to the radius ofgyration of DNA, a 250 bp DNA strand would be within ˜35 nm of the poreentrance, and a 2.5 kbp DNA strand would be within ˜120 nm of the poreentrance. In order to decrease the likelihood that dNMPs are lost insolution, the nanopore could be placed in a well, as described herein.

In some embodiments, both the exonuclease and the nucleic acid aretethered in close proximity to the nanopore. In order to allow forinteraction between the bound species, one of the pair is attached suchthat it has enough mobility to diffuse into contact with the other. Insome cases, one of the exonuclease or template is attached loosely, on arelatively long tether (e.g. a polyethylene glycol chain), and the otheris attached more rigidly near the entrance of the pore. For example, insome embodiments, the exonuclease is bound so that it is held near theentrance to the pore, and the template nucleic acid is attached vialinker molecule that allows it to diffuse into the exonuclease forreaction. Where the template nucleic acid is relatively long, and thedistance between the attachment points of the exonuclease and thetemplate proximate to the nanopore are close, the length and flexibilityof the linker need not be as great.

In another embodiment, the template is anchored on both ends. This tendsto keep the exonuclease close to the nanopore mouth. For example, if thetemplate is dsDNA, then both ends could be biotinylated and fixed to oneor more streptavidins flanking the nanopore. An example of a templateanchored at both ends is shown in FIG. 25.

The attachment of the template can be utilized with a solid statenanopore, a protein nanopore, or a hybrid nanopore. The template DNAstrand could also be attached to a hybrid protein/solid-state nanoporeor to the functionalized edge of a solid-state nanopore. For example, asolid-state nanopore can be surrounded with an annulus of gold or smallgold spheres, and a thiolated DNA template can be used to provideattachment for the template.

Methods for Multiple Pass Sequencing

One aspect of the invention is a method for performing consensusnanopore sequencing of a single molecule of ssDNA. The method allows fora ssDNA molecule to be sequenced repeatedly, significantly improving theaccuracy of nanopore sequencing. The method comprises the followingsteps: Step 1: start with solution of ssDNA to be sequenced, Step 2:attach a linker molecule (e.g. biotin) to 3′ end of the ssDNA, Step 3:Conjugate to a large label (e.g. streptavidin) that cannot pass throughthe nanopore, Step 4: attach a linker molecule to 5′ end of the ssDNA,Step 5: Add labeled ssDNA to cis side of nanopore. Apply potentialdifference across nanopore, which will electrophoretically draw onemolecule of ssDNA at a time through nanopore, Step 6: trans side ofnanopore should contain another large label (that specifically binds tothe linker molecule on 5′ end of ssDNA). Once the ssDNA begins passingthrough the nanopore, this large label attaches to the 5′ end, Step 7:Sequence the ssDNA as it is drawn through the nanopore to the transside, Step 8: When it reaches the end and gets trapped (can be detectedby no change in current), reverse the potential. One can either sequencethe ssDNA backwards, or one can push the ssDNA all the way back to thecis side and start over again, Step 9: When enough consensus sequenceshave been obtained, use standard biochemistry techniques (including pHor temperature changes, or photocleavage) to cleave labels from ssDNAand allow it to pass completely to trans side, Step 10: Start again witha new strand of ssDNA. The method is illustrated in FIG. 26 and FIG. 27.

In some embodiments, the 3′ end could go through the nanopore first. Anysuitable linker molecule that can be attached to the end of ssDNA couldbe used, along with any large particle/protein/molecule that willspecifically attach to this linker and trap the ssDNA in the nanopore.In some cases, instead of using a linker/label to trap the ssDNA, onecould simply hybridize complementary DNA to each end of the ssDNA tomake it double-stranded (single dsDNA cannot pass through the nanopore).This method could be implemented by creating universal adapter sequences(e.g. polyA or polyT tails) at each end of the ssDNA.

Event Driven Detection

One aspect of the invention is a method for determining sequenceinformation about a polymer molecule comprising: (a) obtaining a devicehaving an array of nanopores, each connected to upper and lower fluidregions; wherein the device comprises electronic circuits electricallyconnected to electrodes in either the upper fluid regions or lower fluidregions or both the upper and lower fluid regions; (b) placing a polymermolecule in an upper fluid region; (c) applying a voltage across thenanopore whereby the polymer molecule is translocated through thenanopore; (d) using the electronic circuits to monitor the currentthrough the nanopore over time, wherein the electronic circuits processthe incoming current over time to record events, thereby generatingevent data; and (e) using the event data of step (d) to obtain sequenceinformation about the polymer molecule.

In some cases the events comprise a change in current level above aspecified threshold. In some cases the electronic circuit records theevents, the average current before the events and the average currentafter the events.

In some cases the event data is generated without reference to time. Insome cases a clock circuit is used such that the relative time that theevents occurred is also determined.

In some cases the event data generated by the electronic circuits on thedevice is transmitted from the device for further processing. In somecases the information is transmitted optically.

Base Calling Methods

Nanopore sequencing generally does not achieve single nucleotideresolution, especially in embodiments that might be scaled to acommercially viable DNA sequencing system. Rather, the amplitude ofelectric current passing through the nanopore (which constitutes thesignal) depends on the identity of the several bases that reside in thepore throughout the duration of the current measurement. Thus, ratherthan there being 4 distinct current levels (for A,G,C,T) when the ssDNAtranslocates through the nanopore, there are 4 to the N levels (N=thenumber of bases that affect the current measurement), some of which maybe degenerate (see FIG. 28). Furthermore, the bases residing in thecenter of the nanopore likely affect the current measurement more thanthose near the entrance or exit.

One aspect of the invention is a method for processing information fromnanopore sequencing obtaining improved base calling. In some cases, themethod will enable single base calling from raw data that in unprocessedform cannot call to the level of a single base. The invention involvesdeconvoluting the current measurements in order to achieve singlenucleotide resolution. For example, if one knows that only 3 contiguousbases on the ssDNA strand determine the current measurement at any givetime, then there are 4³=64 possible current levels (some of which mightbe degenerate). One embodiment involves synthetically creating 64different ssDNA strands with all the possible 3-base combinations, andthen pre-calibrating the system by measuring the current blockage levelsfrom each of these ssDNA strands. Subsequent measurements on ssDNA inwhich the sequence is unknown are then compared to this pre-calibrationmeasurement. In an alternative embodiment, the four current levelsassociated with 4 DNA homopolymers (e.g. AAA, GGG, CCC, TTT) aredetermined, allowing the amount by which each position contributes tothe current level (e.g. by comparing AAA to TAA to AAT) to be derived.For example, where it is measured that the nucleotide in the center ofthe nanopore contributes to 75% of the current blockage, the previousnucleotide (−1) contributes 15%, and the subsequent nucleotide (+1)contributes 10%, then a deconvolution can be performed calculate thepredicted current blockage from the various combinations, which can inturn be used to obtain the sequence on an unidentified ssDNA strand bymeasuring its current blockage.

Because the response time of the measurement system (enzyme pluselectrical junction) can be slow in comparison to the single-nucleotiderate through the pore, the measure signal is a convolution of thecurrent perturbation and a impulse function (hereafter called thebase-spread function or “bsf”). Deconvolution of the observed signalwhich arises from convolution with a known kernel in the method of theinvention can be done by, for example, Wiener deconvolution, Janssondeconvolution, or Richardson-Lucy deconvolution.

Basecalling such a signal requires the following steps: deconvolution,peak finding, and peak classification. A fourth optional step which islikely desirable is a quality estimation (“QV” estimation). Peak findingentails finding maximal points in the deconvolved signal which match thecharacteristics of known peaks (i.e. proper amplitude and width). Anexample of such an algorithm is a matched filter or derivative crossingalgorithm. Peak classification can be approached by many differentstatistical classification algorithms such as heuristic decision-treealgorithms, Bayesian networks, hidden Markov models, and conditionalrandom fields.

The application of a deconvolution algorithms generally assumes a knownbsf with constant properties across the signal. The establishment of theform bsf can be identified from control sequence as described above.

Given the nature of single-molecule measurements it is highly likelythat the bsf will vary from trace to trace and even within local regionsof a given trace. This complicates the use of off-the-shelfdeconvolution algorithms. Where the bsf changes on a relatively slowtime scale then a windowed deconvolution can be applied by segmentingthe signal first.

Windowed deconvolution is applied, for example, where we can estimatethe bsf for each window. If we can rely on the kinetics of the signalhaving isolated peaks then the form of the bsf can be estimated byidentifying such peaks in the signal. Alternatively a blinddeconvolution technique can be applied, i.e. optimize the bsf across thewindow until the best contrast is obtained (similar to auto-focus orautomated image restoration algorithms).

In addition, where resequencing is being performed, and the accuracy ofany individual measurement is high, then in some cases, single baseresolution is not required in order to align a measured sequence withthe reference genome, and the known sequence information can be used inconjunction with these methods to improve accuracy. For example, thereference sequence can be convolved with the known bsf and the matchingcan be performed in the convolved space.

When measuring the voltage and setting a threshold (e.g. 2 sigma) forcomparison to a lookup table of all possible sequence context voltages,one might adjust this threshold or the baseline at each position in thetemplate based on slow, global fluctuations (perhaps due to fluctuationsin the power source); or based on a noise model indicating that thistemplate region results in noisier signals; or based on fluctuatingcross-talk noise from neighboring nanopores. An algorithm for using alookup table in this manner is shown in FIG. 29.

One aspect of the invention is a method for determining the sequence ofa polymer having two or more types of monomeric units in a solutioncomprising: (a) actively translocating the polymer through a pore; (b)measuring a property which has a value that varies depending on whetherand which of the two or more a type of monomeric unit is in the pore,wherein the measuring is performed as a function of time, while thepolymer is actively translocating; and (c) determining the sequence ofthe two or more types of monomeric units in the polymer using themeasured property from step (b) by performing a process including thesteps of: (i) deconvolution, (ii) peak finding, and (iii) peakclassification.

In some cases the polymer is a nucleic acid, the monomeric units arenucleotide bases or nucleotide analogs, and the measured property iscurrent. In some cases the deconvolution comprises (a) carrying outmeasurements of current as a function of time on nucleic acids havingknown sequences to produce calibration information, and (b) using thecalibration information perform the deconvolution. In some casesdeconvolution uses a Weiner, Jansson, or Richardson-Levy deconvolution.

In some cases the peak classification is performed by a heuristic treealgorithm, Bayesian network, hidden Markov model, or conditional randomfield. In some embodiments the method further comprises step (iv) ofquality estimation.

In some cases the measurements are on nucleic acids having knownsequences comprising known n-mers. In some cases the known n-mers are3-mers, 4-mers, 5-mers or 6-mers.

In single-molecule nanopore sequencing based on exonuclease release of abase into a nanopore that separates two chambers with a voltage dropbetween them, three metrics include the amplitude of the currentblockage (associated with numerous characteristics of the nucleotide,such as size and charge), the duration of the current blockage(associated with the nucleotide's interaction with the inside of thepore), and the interpulse duration (associated with the dead-time inbetween exonuclease events). One aspect of the invention is algorithmsfor combining information about these three metrics to determine theidentity of a base.

In single-molecule nanopore sequencing based on exonuclease release,generally only one current reading is obtained per nucleotide that flowsthrough the nanopore. Thus, if the probability distribution of currentblockage (likely Gaussian-like) for a nucleotide is highly overlappingwith that of a different nucleotide, then there may be a largeprobability of miscall if only this metric is used. One can combine thisinformation with information from the probability distribution ofcurrent blockage duration (likely exponential-like) for each nucleotide.In one algorithm of the invention, one takes the measurements of currentblockage amplitude and current blockage duration, computes a probabilityof nucleotide-identity for each metric (based on previously calibratedexperiments and determination of the probability distributions), andadds these probabilities in quadrature to obtain an overall probabilityof nucleotide-identity. For example, if P_(A)(duration)=x, andP_(A)(amplitude)=y, then P_(A)(overall)=√{x²+y²}.

Alternatively, one could weight the metrics depending on their relativeimportance or relative uncertainty. Thus, if one placed an importance ofq on pulse duration, then P_(A)(overall)=√{q*x²+(1−q)*y²}. In the caseof an exonuclease chewing up dsDNA, the interpulse duration likelydepends on the sequence context and the secondary structure of the DNA.The measurement of interpulse duration could be added into thequadrature computation, e.g. P_(A)(interpulse duration)=z andP_(A)(overall)=√{x²+y²+z²} or with appropriate weighting.

A second algorithm uses the probability of base-identity obtained fromone metric to alter the probability distribution of a second metric,after which the altered probability distribution the second metric isused to call the base. For example, Base 1 and Base 2 have overlappingcurrent blockage amplitude probability distributions (call them P1 andP2). Once the current blockage duration is measured and compared againstthe probability distribution of the current blockage duration, one cancreate a new current blockage amplitude probability distributions foreach base, call P1′ and P2′. If the current blockage durationmeasurement was more likely to come from Base 1, then P1′ would be widerthan P1, and P2′ would be narrower than P2, but the area under eachdistribution would remain the same. Thus, the overlap between P1′ andP2′ is different from the overlap between P1 and P2. One then uses P1′and P2′ and the current blockage amplitude measurement to identify theunknown nucleotide. In a similar manner, the information from theinterpulse duration measurement could also be used to alter P1′ and P2′and obtain P1″ and P2″.

Dynamic Interventional Nanopore Sequencing

One aspect of the invention involves dynamically reversing the drivingfield in order to obtain repeated reads of the same sequence to improveaccuracy. In embodiments of sequencing in which ssDNA iselectrophoretically drawn through a nanopore (either solid-state orprotein), low inherent base calling accuracy can be a problem. Forexample, if the rate of translocation of each nucleotide through thenanopore follows an exponential distribution, there will be many fasttranslocation events that will lead to low SNR event measurements.Furthermore, the current blockage levels of each of the four nucleotideswill likely have overlapping distributions, leading to the possibilityof miscall errors. A method of real-time re-sequencing of ssDNA regionsin which low accuracy is suspected would greatly improve the overallaccuracy of nanopore sequencing.

Where ssDNA is electrophoretically drawn through a nanopore—from the cischamber to the trans chamber, applying a reverse potential can move thessDNA backwards—from the trans chamber toward the cis chamber. Reversingthe potential in real time when, for example, a suspicious base call ismade can enable an additional measurement of that region of thenucleotide. For example, an algorithm could automatically reverse thepotential if the following events are detected: 1. A very short durationcurrent pulse is detected, which likely has low signal-to-noise, 2. Acurrent pulse's amplitude is in between the peaks of the distributionsfor two different bases, in which case the probability of a miscall ishigh, 3. An unusually long pulse (indicated the possible existence ofhomopolymers, which could lead to deletion or insertion errors), 4. Thetime in between two pulses is unusually long, implying a largelikelihood of a miscall. or 5. There is more noise than usual at thistemplate position (due to a drift in the baseline, due to stochasticcross-talk from neighboring nanopores, or due to sequence context).

The invention involves dynamically controlling the applied potential inorder to enable re-sequencing of low-accuracy regions of the ssDNA. Oneembodiment involves training the basecaller on known ssDNA templates inorder to improve its ability to detect low-accuracy regions.

In some cases, when reversing the potential, the reverse current couldbe measured, in order to measure the sequence in the reverse directionwhile the ssDNA is moving backwards. In addition, when switching thepotential back to its normal sign (i.e. reversing the reversedpotential), one could lower the amplitude of the voltage in order todraw the ssDNA through the nanopore more slowly to enable a higher SNRread of the suspicious nucleotide. In some cases, the potential could bereversed with an amplitude/duration such that only 1 nucleotide isre-sequenced, or more than one nucleotide is resequenced. A flow chartillustrating is method is shown in FIG. 30.

In order to practice dynamic intervention, it is important that thecapacitance of the system be in a suitable range in order to allowreversal of the current at the required frequency. We have determinedthat in some embodiments, where the electrical resistance across thenanopore is about 5 giga-ohms, the capacitance should be less than about3.2 fF in order to have a response time of 0.1 ms. For a resistance of 5giga-ohms and a response time of about 1 ms the capacitance should beless than about 32 fF. For a resistance of 5 giga-ohms and a responsetime of about 10 ms the capacitance should be less than about 320 fF.For a resistance of 5 giga-ohms and a response time of about 0.01 ms thecapacitance should be less than about 0.32 fF. Thus, for use withdynamic intervention, the nanopore structures are produced to have acapacitance that falls in this range or lower. The capacitance of thenanopore structures can be lowered, for example, by controlling thegeometry of the structures that make up the nanopore, and by controllingthe materials that comprise the nanopore structure. In some cases, thehybrid nanostructures described herein can produce lower capacitancenanopore structures by minimizing the amount of or by eliminating thearea of lipid bilayer surrounding the nanopore.

In some embodiments, the capacitance of a nanopore structure comprisinga phospholipid bilayer is lowered by incorporating non-conductivetransmembrane proteins. The transmembrane proteins can have the effectof increasing the thickness of the bilayer, and the increase inthickness can result in a lowering of the capacitance of the bilayer andtherefore the nanopore structure. The non-conductive transmembraneprotein can any suitable protein including plugged nanopore proteins ortransmembrane signaling proteins. The proteins can be fusion proteinshaving some portions that are membrane soluble and other portions thatare water soluble. The relative size of the portions can be controlledto control the properties of the membrane layer.

Magnetic Particles for Control of Polymer Translocation

One aspect of the invention involves the use of magnetic particles thatare associated with the pore or membrane the pore resides in. Themagnetic particle's movement could be controlled by magnetic fields,which would have little effect on the rest of the system, as mostbiologically relevant molecules are not sensitive to magnetic fields.

In one embodiment the magnetic particle is tethered to the nanoporeclose to the entry point of the polymer. Without a magnetic field, thisparticle would be free to float around the polymer, and would not tendto inhibit its motion through the pore (FIG. 31(a)). When a magneticfield is applied the particle is pulled in a direction that results inthe complete or partial plugging the pore, or in pinning of the polymer(FIG. 31(b)).

Similar pore regulations mechanism exist naturally, and have beenreferred to as “Ball and Chain” pore regulators. See, e.g. Jiang et al.Nature, Vol. 417, 523-526, 2002.

In some cases, by controlling the field strength and makeup of thedevice, pinning the biopolymer to the pore can sufficiently slow themovement through the pore. In some cases, a lock step movement can becreated, for example, using a pulsed magnetic field. A pulsed magneticfield may allow the particle to pin-release-pin the biopolymer allowingfor further controlling translocation rates and detection times. Inaddition, the magnetic particle may be used to change the overallelectrical characteristics of the pore, such that one can read out whenthe biopolymer is pinned, and when it is not.

In other embodiments, magnetic particles can exert a force to controlpore characteristics. For example, a magnetic force can cause thenatural pore opening to change in size or shape (FIG. 31(c)). Inaddition, the magnetic particle can influence the shape of the membranethe nanopore is embedded in, thus influencing shape/size of the nanoporeindirectly. (FIG. 31(d)).

EXAMPLES Example 1: Sequencing with Polymerase Enzyme in Nanochannel—SiN

In one embodiment, an array of 256×256 nanochannels, each withapproximate dimensions 100-nm×40-nm×40-nm, are fabricated in a siliconnitride (SIN) substrate using techniques well-known in the art. Whilesurfaces outside the nanochannels are passivated with an inert polymer,such as PEG, the inner surface of each channel is modified withbiotinylated silane using techniques well-known in the art. A φ29 DNApolymerase, modified to have a C- or N-terminal biotin tag, isconjugated to streptavidin. A DNA template, e.g. a cyclic DNA templatesuch as a SMRTbell (Pacific BioSciences) with a primer, is captured bythe polymerase. This streptavidin/polymerase/DNA complex is then loadedonto the nanochannel array at a concentration and for a duration suchthat ˜37% nanochannels contain only a single complex (Poisson loading).The nanochannels are bathed in a solution containing the necessarycomponents for both DNA synthesis by the polymerase (e.g. metal ion,four nucleotide analogs, etc.) and for current flow through the channel(e.g. salt). A voltage of ˜100-800 mV is applied across thenanochannels. The nucleotide analogs are labeled at theirterminal-phosphate with a latex particle. Each of the four analogstypes, corresponding to the four nucleotides, is labeled with adifferent sized latex particle (e.g. 10-nm, 15-nm, 20-nm, 25-nmdiameters). While the cognate nucleotide is being incorporated by thepolymerase into the growing strand complementary to the DNA template,the label alters the current flowing through the nanochannel. Each typeof label alters the current in a way distinct from the other labels, andthus the identity of the incorporated base is determined. As a naturalpart of the incorporation process, the polymerase cleaves the label fromthe nucleotide, allowing the growing DNA strand to be label-free.

Example 2: Sequencing with Polymerase Enzyme in Nanochannel—SiOx

In another embodiment, an array of 256×256 nanochannels, each withapproximate dimensions 100-nm×40-nm×40-nm, are fabricated in a siliconoxide (SiOx) substrate using techniques well-known in the art. Whilesurfaces outside the nanochannels are passivated with an inert polymer,such as PEG, the inner surface of each channel is modified withbiotinilated silane using techniques well-known in the art. A φ29 DNApolymerase, modified to have a C- or N-terminal biotin tag, isconjugated to streptavidin. A DNA template, e.g. a cyclic DNA templatesuch as a SMRTbell (Pacific BioSciences) with a primer, is captured bythe polymerase. This streptavidin/polymerase/DNA complex is then loadedonto the nanochannel array at a concentration and for a duration suchthat ˜37% nanochannels contain only a single complex (Poisson loading).The nanochannels are bathed in a solution containing the necessarycomponents for both DNA synthesis by the polymerase (e.g. metal ion,four nucleotide analogs, etc.) and for current flow through the channel(e.g. salt). A voltage of ˜100-800 mV is applied across thenanochannels. The nucleotide analogs are labeled at theirterminal-phosphate with a latex particle. Each of the four analogstypes, corresponding to the four nucleotides, is labeled with adifferent sized latex particle (e.g. 10-nm, 15-nm, 20-nm, 25-nmdiameters). While the cognate nucleotide is being incorporated by thepolymerase into the growing strand complementary to the DNA template,the label alters the current flowing through the nanochannel. Each typeof label alters the current in a way distinct from the other labels, andthus the identity of the incorporated base is determined. As a naturalpart of the incorporation process, the polymerase cleaves the label fromthe nucleotide, allowing the growing DNA strand to be label-free.

Example 3: Sequencing with Polymerase Enzyme in Nanochannel—PolymericSubstrate

In another embodiment, an array of 256×256 nanochannels, each withapproximate dimensions 100-nm×40-nm×40-nm, are fabricated in a polymericsubstrate with backbone containing thiol-acrylate using techniqueswell-known in the art. While surfaces outside the nanochannels arepassivated with an inert polymer, such as PEG, the inner surface of eachchannel is modified with biotinylated maleimide using techniqueswell-known in the art. A φ29 DNA polymerase, modified to have a C- orN-terminal biotin tag, is conjugated to streptavidin. A DNA template,e.g. a cyclic DNA template such as a SMRTbell (Pacific BioSciences) witha primer, is captured by the polymerase. Thisstreptavidin/polymerase/DNA complex is then loaded onto the nanochannelarray at a concentration and for a duration such that ˜37% nanochannelscontain only a single complex (Poisson loading). The nanochannels arebathed in a solution containing the necessary components for both DNAsynthesis by the polymerase (e.g. metal ion, four nucleotide analogs,etc.) and for current flow through the channel (e.g. salt). A voltage of˜100-800 mV is applied across the nanochannels. The nucleotide analogsare labeled at their terminal-phosphate with a latex particle. Each ofthe four analogs types, corresponding to the four nucleotides, islabeled with a different sized latex particle (e.g. 10-nm, 15-nm, 20-nm,25-nm diameters). While the cognate nucleotide is being incorporated bythe polymerase into the growing strand complementary to the DNAtemplate, the label alters the current flowing through the nanochannel.Each type of label alters the current in a way distinct from the otherlabels, and thus the identity of the incorporated base is determined. Asa natural part of the incorporation process, the polymerase cleaves thelabel from the nucleotide, allowing the growing DNA strand to belabel-free.

Example 4: Sequencing with Polymerase Enzyme in Nanochannel—SiN andSilica Particles on Nucleotides

In another embodiment, an array of 256×256 nanochannels, each withapproximate dimensions 100-nm×40-nm×40-nm, are fabricated in a SiNsubstrate using techniques well-known in the art. While surfaces outsidethe nanochannels are passivated with an inert polymer, such as PEG, theinner surface of each channel is modified with biotinilated silane usingtechniques well-known in the art. A φ29 DNA polymerase, modified to havea C- or N-terminal biotin tag, is conjugated to streptavidin. A DNAtemplate, e.g. a cyclic DNA template such as a SMRTbell (PacificBioSciences) with a primer, is captured by the polymerase. Thisstreptavidin/polymerase/DNA complex is then loaded onto the nanochannelarray at a concentration and for a duration such that ˜37% nanochannelscontain only a single complex (Poisson loading). The nanochannels arebathed in a solution containing the necessary components for both DNAsynthesis by the polymerase (e.g. metal ion, four nucleotide analogs,etc.) and for current flow through the channel (e.g. salt). A voltage of˜100-800 mV is applied across the nanochannels. The nucleotide analogsare labeled at their terminal-phosphate with a latex particle. Each ofthe four analogs types, corresponding to the four nucleotides, islabeled with a different sized silica particle (e.g. 10-nm, 15-nm,20-nm, 25-nm diameters). While the cognate nucleotide is beingincorporated by the polymerase into the growing strand complementary tothe DNA template, the label alters the current flowing through thenanochannel. Each type of label alters the current in a way distinctfrom the other labels, and thus the identity of the incorporated base isdetermined. As a natural part of the incorporation process, thepolymerase cleaves the label from the nucleotide, allowing the growingDNA strand to be label-free.

Example 5: Simulation Demonstrating Base Calling Using SignalsCharacteristic of More than One Base to Call Bases at Single BaseResolution

A simulation was performed that demonstrated the ability to determinethe identity of a DNA sequence as it translocates through a nanopore,given that the resolution of the measurement system is >1 nucleotide(i.e. the measurement is influenced by the identity and position of anumber of nucleotides, e.g. 5 that reside within the nanopore at anygiven moment). The algorithm uses a lookup table as shown in FIG. 29.The algorithm is for use with a lookup table created for the signalsyielded by every possible permutation of the several bases that affectthe measurement. Some of these signals will be degenerate with oneanother within the error of the measurement. Given a measurement, thisalgorithm compares the signal with the lookup table and keeps track ofall the possible 5-mers that could account for the measurement.

After each single-nucleotide translocation through the nanopore, thealgorithm looks up the possible 5-mers for that measurement and thenthrows away all the possibilities from the previous measurement that arenot consistent with the most recent measurement. Thus, even if the firstmeasurement yielded many possible sequences, it is likely that afterseveral measurements there will only be one or a few possible sequencesthat are consistent with all the measurements (this will depend on thedistribution of voltages in the lookup table and on the accuracy of themeasurements).

The above description is intended to be illustrative and notrestrictive. It readily should be apparent to one skilled in the artthat various embodiments and-modifications may be made to the inventiondisclosed in this application without departing from the scope andspirit of the invention. The scope of the invention should, therefore,be determined not with reference to the above description, but shouldinstead be determined with reference to the appended claims, along withthe full scope of equivalents to which such claims are entitled. Allpublications mentioned herein are cited for the purpose of describingand disclosing reagents, methodologies and concepts that may be used inconnection with the present invention. Nothing herein is to be construedas an admission that these references are prior art in relation to theinventions described herein. Throughout the disclosure various patents,patent applications and publications are referenced. Unless otherwiseindicated, each is incorporated by reference in its entirety for allpurposes.

What is claimed is:
 1. A method for sequencing a nucleic acid templatecomprising: providing a substrate having an upper solution above thesubstrate and a lower solution below the substrate, the substratecomprising a nanopore connecting the upper solution and lower solution,the nanopore sized to pass a single strand of a nucleic acid; providinga voltage across the nanopore to produce a measurable current flowthrough the nanopore; controlling the rate of translocation of a singlestranded portion of the nucleic acid template through the nanopore witha translocating enzyme that is associated with the nucleic acid templateunder reaction conditions whereby the translocating enzyme and thereaction conditions are selected such that the translocating enzymeexhibits two kinetic steps wherein each of the kinetic steps has a rateconstant, and the ratio of the rate constants of the kinetic steps isfrom 10:1 to 1:10; measuring the current through the nanopore over timeas the nucleic acid template is translated through the nanopore; anddetermining the sequence of a portion of the nucleic acid template as ittranslates through the nanopore using the measured current over time. 2.The method of claim 1 wherein the translocating enzyme comprises apolymerase, an exonuclease, or a helicase.
 3. The method of claim 1wherein the translocating enzyme comprises a DNA helicase or an RNAhelicase.
 4. The method of claim 1 wherein the translocating enzymecomprises phi29 DNA polymerase, T7 DNA polymerase, T4 DNA polymerase, E.coli DNA pol 1, Klenow fragment, T7 RNA polymerase, or E. coli RNApolymerase.
 5. The method of claim 1 wherein the translocating enzymecomprises a viral genome packaging motor or an ATP-dependent chromatinremodeling complex.
 6. The method of claim 1 wherein the two kineticsteps are selected from a group consisting of enzyme isomerization,nucleotide incorporation, and product release.
 7. The method of claim 1wherein the two kinetic steps are template translocation and nucleotidebinding.
 8. The method of claim 1 wherein the rate for one of thekinetic steps is less than 100 per second.
 9. The method of claim 1wherein the rate for one of the kinetic steps is between 0.5 per secondand 60 per second.
 10. The method of claim 1 wherein the reactionconditions comprise one or more of metal cofactor concentration, pH,temperature, an enzyme activity modulator, D₂O, an organic solvent, andbuffer.
 11. The method of claim 1 wherein the nucleic acid templatecomprises DNA.
 12. The method of claim 1 wherein the substrate comprisesan array of nanopores.
 13. The method of claim 1 wherein the ratio ofthe rate constants is from 1:5 and 5:1.
 14. The method of claim 1wherein the ratio of the rate constants is from 1:2 to 2:1.
 15. Themethod of claim 1 wherein the nanopore comprises a solid state nanopore.16. The method of claim 1 wherein the nanopore comprises a proteinnanopore in a lipid bilayer membrane.
 17. The method of claim 1 whereindetermining the sequence comprises comparing the measured currentthrough the nanopore over time with known current over time values fornucleic acid n-mers comprising 3-mers, 4-mers, 5-mers, or 6-mers. 18.The method of claim 17 wherein the n-mers comprise 3-mers.